Specific delivery of agrochemicals

ABSTRACT

Described is the specific delivery of agrochemicals to plants. More specifically, a targeting agent has at least one binding domain that specifically binds to a binding site on an intact living plant. Such binding domains include a peptide having 4 framework regions and 3 complementary determining regions, or fragment(s) thereof, wherein the binding domains bind or retain a carrier onto a plant. Described are binding domains that specifically bind trichomes, stomata, cuticle, lenticels, thorns, spines, root hairs, or wax layer. Further described are methods for delivering agrochemicals to a plant, for depositing agrochemicals on a plant, and for retaining the agrochemicals on a plant, using targeting agents comprising the binding domains, and to methods for protecting a plant against stress or controlling plant growth. Also, described are methods for manufacturing a specifically targeting agrochemical carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/017,637, filed Sep. 4, 2013, which is a continuation of U.S.patent application Ser. No. 13/081,435, filed Apr. 6, 2011, now U.S.Pat. No. 8,598,081, issued Dec. 3, 2013, which claims the benefit under35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.61/341,930, filed Apr. 6, 2010, and under Article 8 of the PCT toEuropean Patent Application Serial No. EP 10159100.6, filed Apr. 6,2010, the disclosure of each of which is hereby incorporated herein inits entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The disclosure relates to specific delivery of agrochemicals to plants.More specifically, it relates to a composition, essentially consistingof a targeting agent comprising at least one binding domain thatspecifically binds to a binding site on an intact living plant and anagrochemical or a combination of agrochemicals. The disclosure relatesfurther to a binding domain that specifically binds the binding site onan intact living plant. More specifically, it relates to binding domainscomprising an amino acid sequence that comprises four framework regionsand three complementary determining regions, or any suitable fragmentthereof, wherein the binding domains are capable to bind or retain acarrier onto a plant. In one embodiment, it relates to binding domainswhich specifically bind trichomes, stomata, cuticle, lenticels, thorns,spines, root hairs, or wax layer. It relates further to a method fordelivery of agrochemicals to a plant, for improving the deposition ofagrochemicals on a plant, and for retaining the agrochemicals on aplant, using targeting agents comprising the binding domains, and to amethod for protecting a plant against biotic or abiotic stress orcontrolling plant growth using the same. Also, it relates to a methodfor manufacturing a specifically targeting agrochemical carrier.

BACKGROUND

For many years, horticulturist and agronomist have applied chemicals forweed control, plant protection and plant growth regulation by sprayingthe fields. For compositions that need to be applied on the plant, e.g.,on the foliage, only a small part of the composition is bound to andretained on the part of the plant where it can exert its biologicalactivity as large amounts are not adhering to the plant surface and arelost by drip-off or washed away by rain. Apart from giving rise toreduced efficacy of the chemical, losses of chemicals into the soil dueto dripping off the plant while spraying or due to wash-out duringrainfall may result in groundwater contamination, environmental damage,loss of biodiversity, and human and animal health consequences.

Several researchers have tried to solve this problem by applying slowrelease particles to the plant that stick to the leaves and releasetheir content over a certain period of time. U.S. Pat. No. 6,180,141describes composite gel microparticles that can be used to deliverplant-protection active principles. WO 2005102045 describes compositionscomprising at least one phytoactive compound and an encapsulatingadjuvant, wherein the adjuvant comprises a fungal cell or a fragmentthereof. U.S. 20070280981 describes carrier granules, coated with alipophilic tackifier on the surface, wherein the carrier granule adheresto the surface of plants, grasses and weeds.

Those microparticles, intended for the delivery of agrochemicals, arecharacterized by the fact that they stick to the plant by rather weak,aspecific interactions, such as a lipophilic interaction. Although thismay have advantages compared with the normal spraying, the efficacy ofsuch delivery method is limited, and the particles may be non-optimallydistributed over the leaf, or washed away under naturally variableclimatological conditions, before the release of the compound iscompleted. For a specific distribution and efficient retention of themicroparticles, a specific, strongly binding molecule is needed that canassure that the carrier sticks to the plant till its content iscompletely delivered.

Cellulose binding domains (CBDs) have been described as useful agentsfor attachment of molecular species to cellulose (U.S. Pat. Nos.5,738,984 and 6,124,117). Indeed, as cotton is made up of 90% cellulose,CBDs have proved useful for delivery of so called “benefit agents” ontocotton fabrics, as is disclosed in WO9800500 where direct fusionsbetween a CBD and an enzyme were used utilizing the affinity of the CBDto bind to cotton fabric. The use of similar multifunctional fusionproteins for delivery of encapsulated benefit agents was claimed inWO03031477, wherein the multifunctional fusion proteins consist of afirst binding domain which is a carbohydrate binding domain and a secondbinding domain, wherein either the first binding domain or the secondbinding domain can bind to a microparticle. WO03031477 is exemplifiedusing a bifunctional fusion protein consisting of a CBD and an anti-RR6antibody fragment binding to a microparticle, which complex is depositedonto cotton treads or cut grass. However, the use of suchmultifunctional fusion proteins for delivery of encapsulated benefitagents suffers from a number of serious drawbacks. First, althoughcellulose is a major component of plant cell walls and about 33% of allplant matter consists of cellulose, cellulose is, in intact livingplants, shielded off from the outside environment by the plant cuticle,formed by cutin and waxes, which is an impermeable barrier with whichplant cell walls are covered, making cellulose poorly accessible forbinding by CBDs. Secondly, effective delivery of an encapsulated benefitagent to the plant requires simultaneous binding of the first bindingdomain to the plant and the second binding domain to the microparticle.As the likelihood of both binding events occurring is determined by adelicate equilibrium between the molar concentrations of the bindingdomains and their target molecules and the molar concentration of thebound complex, it is highly unlikely that sufficient multifunctionalfusion proteins are present in solution to enable such simultaneousbinding. Moreover, the equilibrium of a binding event is stronglyinfluenced by environmental parameters such as temperature and pH, forwhich the optimal conditions may be considerably different for each ofthe binding domains. Therefore, it is highly unlikely that suchsimultaneous binding of two binding domains of such multifunctionalfusion protein would result in a sufficiently strong binding that wouldretain an encapsulated benefit agent to a plant. Thirdly, althoughbinding of a CBD is to a certain extent specific for cellulose, using amultifunctional fusion protein in which CBD should bind to the plant isto be considered as a generic binding approach, as all plants containcellulose, and is therefore similar to aspecific sticking withtackifiers or stickers. A targeted approach in which specific binding ofa binding domain would allow discrimination between binding to one plantspecies versus another would be of considerably higher value. WO03031477also suggests, without further exemplification, that other binders tocarbohydrates or polysaccharides can be used to generate fusion proteinsto deposit microparticles onto living organisms. However, neitherbinding domains other than CBDs, nor binding domains binding to intactliving plants were disclosed in WO03031477.

Molecules that are well known for their specificity and high affinity toparticular targets are antibodies. Antibodies can be generated against abroad variety of targets, and antibodies that were generated to studyplant cell wall architecture and dynamics have been described to bindspecifically to particular plant constituents, predominantlyconstituents of the plant cell wall (Penell et al., 1989; Jones et al.,1997; Willats et al., 1998; Willats and Knox, 1999; Willats et al.,2001). However, it is unclear whether any of the plant cell wallconstituents to which the antibodies have been generated, would bedirectly accessible for an antibody from the outside environment.Moreover, antibodies are by their very nature as components of theadaptive immune system construed such that they bind their targets underphysiological conditions, including tightly regulated pH, temperature,and blood's normal osmolarity range. Should one consider to useantibodies for targeted delivery of agrochemicals, the antibodies shouldnot only be capable of binding their target on an intact living plant inan agrochemical formulation, for which physicochemical characteristicsdeviate substantially from physiological conditions, they should also becapable to bind strongly enough to retain a carrier onto a plant. Forneither of the plant-binding antibodies earlier described, either ofthese two crucial characteristics have been demonstrated.

The variable domains of camelid heavy chain antibodies (VHH) are aparticularly interesting type of antibody fragments, as they are small,15 kDa single-chain proteins, which can be selected for displaying highaffinity for their targets. Also, by their nature as small single-chainmolecules, VHH are easy to produce and have superior stabilitycharacteristics over conventional antibodies. However, so far, noplant-binding VHH have been described. Moreover, although VHH that arecovalently linked to a solid resin particle have been shown to maintainfunctionality in the sense that they are able to capture antigen from asolution (WO 0144301), it has not been shown, nor can it be expected,that the affinity of VHH for its target is sufficient to retain acarrier onto a solid plant surface.

DISCLOSURE

We have isolated binding domains, more specifically binding domainscomprising an amino acid sequence (or “peptide”) that comprises fourframework regions (FR) and three complementary determining regions (CDR)(FR and CDR definitions according to Kabat), wherein the binding domainsare capable to bind a binding site on an intact living plant and,surprisingly, in doing so, are capable of retaining an agrochemical or acarrier containing an agrochemical to the plant. In certain embodiments,the binding domains remain stable and retain their binding capacityunder harsh conditions, such as variable temperature, pH, saltconcentration, availability of water or moisture. In some embodiments,the binding domains remain stable and retain their binding capacity inan agrochemical formulation. Binding domains comprising four FRs andthree CDRs, preferably in a sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, areknown to the person skilled in the art and have been described, as anon-limiting example in Wesolowski et al. (2009). In certainembodiments, the binding domains are derived from camelid antibodies,preferably, from heavy chain camelid antibodies, devoid of light chains,such as variable domains of heavy chain camelid antibodies (VHH).

Targeting agents comprising these binding domains, can retainagrochemicals specifically to binding sites on the plant or plant partsand can be used to deliver and retain agrochemicals to the plant,preferably to the intact living plant, wherein the binding domainscomprised in such targeting agents specifically bind to binding sites onthe plant, where the agrochemicals can exert their activity.Agrochemical compositions comprising at least one targeting agent and anagrochemical, preferably bound on or comprised in a carrier, may besuitable to allow the use of a reduced dose of the agrochemical and/orreduction of the frequency of application of the agrochemical, comprisedin such composition whilst maintaining its overall efficacy. Moreover,when comprised in a composition hereof, the agrochemical may exert itsactivity over a longer period of time, eventually resulting in lessagrochemical being lost and contaminating the environment; also, byapplying an agrochemical in a composition hereof, it is possible tointroduce specificity into the activity of the agrochemical that isotherwise not present.

Provided is a specific delivery method for agrochemicals in which theagrochemical is delivered or deposited on or near its site of action onan intact living plant utilizing a binding domain that can bindspecifically and strongly to the intact living plant, and is able toretain an agrochemical or a carrier containing the agrochemical onto theplant.

A first aspect, provided is a binding domain capable to bind at leastone binding site on an intact living plant. An “intact living plant,” asused herein, means a plant as it grows, whether it grows in soil, inwater or in artificial substrate, and whether it grows in the field, ina greenhouse, in a yard, in a garden, in a pot or in hydroponic culturesystems. An intact living plant preferably comprises all plant parts(roots, stem, branches, leaves, needles, thorns, flowers, seeds . . . )that are normally present on such plant in nature, although some plantparts, such as, e.g., flowers, may be absent during certain periods inthe plant's life cycle. An intact living plant excludes plant parts thathave been removed from the plant, such as leaves and flowers that havebeen cut and separated from the plant. However, an intact living plantincludes plants that have been damaged by normal natural events such asdamage by weather (such as, but not limited to wind, rain, or hail), byanimals (whether by animals feeding on the plants or by animalstrampling on the plants), by plant pests (such as, but not limited toinsects, nematodes and fungi), or damage caused by agricultural practicesuch as, but not limited to pruning, harvesting of fruit, or harvestingof flowers. Plants include gymnosperms and angiosperms, monocotyledonsand dicotyledons, trees, fruit trees, field and vegetable crops andornamental species. As a non-limiting example, the plants can be cedars,cypresses, firs, junipers, larches, pines, redwoods, spruces, yews,gingko, oilpalm, rubber tree, oak, beech, corn, cotton, soybean, wheat,rice, barley, rye, sorghum, millet, rapeseed, beans, peas, peanuts,sunflower, potato, tomato, sugarcane, sugarbeet, cassava, tobacco,banana, apple, orange, lemon, olive, pineapple, avocado, vines, lettuce,cabbage, carrot, eggplant, pepper, melon, rose, lilies, chrysanthemum,grass-like weeds, or broadleaved weeds.

A “binding site,” as used herein, means a molecular structure orcompound, such as a protein, a (poly)peptide, a (poly)saccharide, aglycoprotein, a lipoprotein, a fatty acid, a lipid or a nucleic acid ora particular region in such molecular structure or compound or aparticular conformation of such molecular structure or compound, or acombination or complex of such molecular structures or compounds. Incertain embodiments, the binding site comprises at least one antigen.“Antigen,” as used herein, means a molecule capable of eliciting animmune response in an animal. In certain embodiments, the binding siteis comprised in a plant structure such as a trichome, stomata,lenticels, thorns, spines, root hairs, cuticle or wax layer. Even morepreferably, the binding site is comprised in a plant structure such as atrichome, stomata or cuticle. The binding site may be unique for oneparticular plant structure, or it may be more generally comprised inmore than one plant structure. In certain embodiments, the binding siteis present on a particular part of the plant, such as the leaves, stems,roots, fruits, cones, flowers, bulbs or tubers. Even more preferably,the binding site is present on the surface of such particular part ofthe plant, meaning that the binding site is present at, for example, theleaf surface, the stem surface, the root surface, the fruit surface, thecone surface, the flower surface, the bulb surface or the tuber surface.The binding site may be unique for one particular plant part, or it maybe more generally present on more than one plant part.

A “binding domain,” as used herein, means the whole or part of aproteinaceous (protein, protein-like or protein containing) moleculethat is capable of binding using specific intermolecular interactions toa target molecule. A binding domain can be a naturally occurringmolecule, e.g., fibronectin, it can be derived from a naturallyoccurring molecule, e.g., from components of the innate or adaptiveimmune system, or it can be entirely artificially designed. A bindingdomain can be immunoglobulin-based or it can be based on domains presentin proteins, including but not limited to microbial proteins, proteaseinhibitors, toxins, fibronectin, lipocalins, single-chain antiparallelcoiled coil proteins or repeat motif proteins. Non-limiting examples ofsuch binding domains are carbohydrate binding domains (CBD) (Blake etal, 2006), heavy chain antibodies (hcAb), single domain antibodies(sdAb), minibodies (Tramontano et al., 1994), the variable domain ofcamelid heavy chain antibodies (VHH), the variable domain of the newantigen receptors (VNAR), affibodies (Nygren et al., 2008), alphabodies(WO2010066740), designed ankyrin-repeat domains (DARPins) (Stumpp etal., 2008), anticalins (Skerra et al., 2008), knottins (Kolmar et al.,2008) and engineered CH2 domains (nanoantibodies; Dimitrov, 2009).Preferably, the binding domain consists of a single polypeptide chainand is not post-translationally modified. More preferably, the bindingdomain is not a CBD. Even more preferably, the binding domain is derivedfrom an innate or adaptive immune system, preferably from a protein ofan innate or adaptive immune system. Still more preferably, the bindingdomain is derived from an immunoglobulin. Most preferably, the bindingdomain comprises four framework regions and three complementarydetermining regions, or any suitable fragment thereof (which will thenusually contain at least some of the amino acid residues that form atleast one of the complementary determining regions). Preferably, abinding domain is easy to produce at high yield, preferably in amicrobial recombinant expression system, and convenient to isolateand/or purify subsequently. Also preferably, a binding domain is stable,both during storage and during utilization, meaning that the integrityof the binding domain is maintained under storage and/or utilizationconditions, which may include elevated temperatures, freeze-thaw cycles,changes in pH or in ionic strength, UV-irradiation, presence of harmfulchemicals and the like. More preferably, the binding domain is stable inan agrochemical formulation. An “agrochemical formulation” as usedherein means a composition for agrochemical use, as further defined,comprising at least one active substance, optionally with one or moreadditives favoring optimal dispersion, atomization, deposition, leafwetting, distribution, retention and/or uptake of agrochemicals. As anon-limiting example, such additives are diluents, solvents, adjuvants,surfactants, wetting agents, spreading agents, oils, stickers,thickeners, penetrants, buffering agents, acidifiers, anti-settlingagents, anti-freeze agents, photo-protectors, defoaming agents, biocidesand/or drift control agents. Most preferably, the binding domain remainsstable in an agrochemical formulation when stored at ambient temperaturefor a period of two years or when stored at 54° C. for a period of twoweeks. Preferably, the binding domain is selected from the groupconsisting of DARPins, knottins, alphabodies and VHH. More preferably,the binding domain is selected from the group consisting of alphabodiesand VHH. Most preferably, the binding domain is a VHH.

Binding of the binding domain to the binding site or to an antigencomprised in the binding site occurs with high affinity. Thedissociation constant is commonly used to describe the affinity betweena binding domain and its target molecule. Preferably, the dissociationconstant of the binding between the binding domain and the targetmolecule comprised in the binding site is lower than10⁻⁵ M, morepreferably, the dissociation constant is lower than 10⁻⁶ M, even morepreferably, the dissociation constant is lower than 10⁻⁷ M, mostpreferably, the dissociation constant is lower than 10⁻⁸ M. Preferably,binding of the binding domain to the binding site is specific, meaningthat the binding domain binds to the binding site only if the targetmolecule is present in the binding site and that the binding domain doesnot bind, or binds with much lower affinity, to a binding site lackingthe target molecule. Specificity of binding of a binding domain can beanalyzed by methods such as ELISA, as described in Example 2, in whichthe binding of the binding domain to its target molecule is comparedwith the binding of the binding domain to an unrelated molecule and withaspecific sticking of the binding domain to the reaction vessel.Specificity can also be expressed as the difference in affinity of abinding domain for its target molecule versus the affinity for anunrelated molecule. Preferably, the ratio of the affinity of the bindingdomain for its target molecule versus its affinity for an unrelatedmolecule is larger than 10, more preferably, the ratio is larger than20, most preferably, the ratio is larger than 100. Binding of thebinding domain can be specific for one particular plant structure,meaning that the binding site, comprised in such plant structure, is notor to a much lesser extent present in other plant structures; or thebinding can be more general to more than one plant structure, if thebinding site is present in more than one plant structure. Binding of thebinding domain can be specific for one particular plant part, meaningthat the binding site, present in or on such plant part, possiblycomprised in a plant structure on such plant part, is not or to a muchlesser extent present in other plant parts; or the binding can be moregeneral to more than one plant part, if the binding site is present inmore than one plant part. Binding of the binding domain can be specificfor one particular plant species, meaning that the binding site, presentin or on such plant species, is not or to a much lesser extent presentin other plant species; or the binding can be more general to more thanone plant species, if the binding site is present in more than one plantspecies. Binding of the binding domain can be specific for oneparticular plant genus, meaning that the binding site, present in or onsuch plant genus, is not or to a much lesser extent present in otherplant genera; or the binding can be more general to more than one plantgenus, if the binding site is present in more than one plant genus.Binding of the binding domain can be specific for one particular growthstage of the plant, meaning that the binding site, present in or on suchplant at a particular growth stage, is not or to a much lesser extentpresent in the plant at another growth stage; or the binding can be moregeneral to more than one plant growth stage, if the binding site ispresent in more than one plant growth stage. All types of bindingspecificity of the binding domains may have their specific use, as willbe explained below.

Preferably, the binding of the binding domain to the binding site isstill functional under harsh conditions, such as low or hightemperature, low or high pH, low or high ionic strength, UV-irradiation,low availability of water, presence of denaturing chemicals or the like.In one embodiment, the harsh conditions are defined by a pH range from 4to 9, more preferably, by a pH range from 3 to 10, even more preferably,by a pH range from 2 to 10, most preferably, by a pH range from 1 to 11.In another embodiment, the harsh conditions are defined by a temperaturerange from 4-50° C., more preferably, a temperature range from 0-55° C.,even more preferably, a temperature range from 0-60° C. In anotherembodiment, the harsh conditions are defined by the presence of anagrochemical formulation as defined above.

Preferably, the binding of the binding domain to the binding site isstrong enough to bind, more preferably, to retain, a carrier to thebinding site; depending on the size of the carrier and on the affinityof the binding domain, one or more binding domains may bind to one ormore binding sites and cooperate such that the resulting avidity of thebinding domains for the binding site(s) ensures strong binding of thecarrier, preferably retaining the carrier, onto the plant. A “carrier,”as used herein, means any solid, semi-solid or liquid carrier in oron(to) which an active substance can be suitably incorporated, included,immobilized, adsorbed, absorbed, bound, encapsulated, embedded,attached, or comprised. Non-limiting examples of such carriers includenanocapsules, microcapsules, nanospheres, microspheres, nanoparticles,microparticles, liposomes, vesicles, beads, a gel, weak ionic resinparticles, liposomes, cochleate delivery vehicles, small granules,granulates, nano-tubes, bucky-balls, water droplets that are part of anwater-in-oil emulsion, oil droplets that are part of an oil-in-wateremulsion, organic materials such as cork, wood or other plant-derivedmaterials (e.g., in the form of seed shells, wood chips, pulp, spheres,beads, sheets or any other suitable form), paper or cardboard, inorganicmaterials such as talc, clay, microcrystalline cellulose, silica,alumina, silicates and zeolites, or even microbial cells (such as yeastcells) or suitable fractions or fragments thereof (as further describedherein). “Retain” as used herein means that the binding force resultingfrom the affinity or avidity of either one single binding domain or acombination of two or more binding domains for its or their binding siteis larger than the combined force and torque imposed by the gravity ofthe carrier, the force and torque, if any, caused by the flow or dripoff of a sprayed agrochemical solution and the force and torque, if any,imposed by shear forces caused by one or more external factors. In oneembodiment, the external factor is rain, irrigation, snow, hail or wind.One particular advantage of binding a carrier by specific binding overaspecific binding is that specific binding is more resistant to externalshear forces applied to the carrier (Cozens-Roberts et al., 1990).

Preferably, a binding domain hereof binds to a binding site, or to anantigen comprised in such binding site, present in or on one or moreparticular parts of the intact living plant. Preferably, the parts ofthe intact living plant are selected from the group consisting ofleaves, stem, roots, fruits, cones, flowers, bulbs or tubers. Morepreferably, the parts of the intact living plant are selected from thegroup consisting of leaves, stem or roots. Preferably, a binding domainhereof binds to a binding site, or to an antigen comprised in suchbinding site, on the surface of the intact living plant. A “surface,” asused herein, can be any surface as it occurs on the intact living plant;or on one or more parts of the intact living plant, however, it excludeshistological plant preparations. Preferably, the surface of the intactliving plant is the surface of a part of the intact living plant,selected from the group consisting of leaf surface, stem surface, rootsurface, fruit surface, cone surface, flower surface, bulb surface ortuber surface; even more preferably, the surface of the intact livingplant is the surface of a part of the intact living plant, selected fromthe group consisting of root surface, stem surface and leaf surface.

Preferably, a binding domain hereof binds to a binding site, or to anantigen comprised in such binding site, in or on a particular structureof the intact living plant or in or on a particular structure of aparticular part of the intact living plant; more preferably, in or on aparticular structure involved or implicated to be involved in transportof nutrients, agrochemicals or other chemicals into the plant and/orinvolved or implicated to be involved in plant defense. Preferably, theparticular structure is selected from the group consisting of trichomes,stomata, lenticels, thorns, spines, root hairs, cuticle and wax layer,even more preferably, the particular structure is selected from thegroup consisting of trichomes, stomata and cuticle. In one embodiment,the binding domain is binding to a binding site, or to an antigencomprised in such binding site, in or on plant trichomes. Planttrichomes are known to the person skilled in the art, and include, butare not limited to glandular trichomes and leaf hairs. Plant trichomesare active in plant defense (Lai et al, 2000), but especiallynon-glandular trichomes are also cited as possible targets for infection(Calo et al., 2006). Trichomes, including glandular trichomes, are alsoimplicated in the transport of polar compounds across plant cuticlesinto the plant (Schreiber, 2005). This makes trichomes an ideal targetfor delivery of agrochemicals, either by enhancing the natural defenseor by concentrating agrochemicals at the site of attack or by improveddelivery of (polar) agrochemicals into the plant. In another embodiment,the binding domain binds to a binding site, or to an antigen comprisedin such binding site, in or on stomata. Stomata are essential to allowCO₂ to diffuse into the plant and to minimize water loss. Stomata arealso used as a major entry site for pathogens, especially microbes(Underwood et al. 2007). “Microbes,” as used herein, means bacteria,viruses, fungi and the like. Moreover they are directly implicated inplant defense via specific signaling pathways allowing the plant toclose stomata upon microbial infection (Melotto et al., 2006). In yetanother embodiment, the binding domain binds to a binding site, or to anantigen comprised in such binding site, in or on root hairs. Root hairsare known to be important for microbial attachment to and colonizationof plants (Gage, 2004; Laus et al., 2005) and are, therefore, animportant target for the delivery of agrochemicals. In anotherembodiment, the binding domain binds to a binding site, or an antigencomprised in such binding site, in or on plant cuticle. Plant cuticlesare known to be important for microbial attachment to and colonizationof plants and to play an important role in delivery and deposition oflipophilic agrochemicals into the plant (Schreiber, 2005) and aretherefore an important target for the delivery and deposition ofagrochemicals.

In one embodiment, the binding domain hereof is binding gum arabic. Inanother embodiment, the binding domain is binding lectins, lectin-likedomains, extensins, or extensin-like domains; more preferably, thebinding domain is binding potato lectin. Preferably, the binding domaincomprises four framework regions and three complementary determiningregions, or any suitable fragment thereof (which will then usuallycontain at least some of the amino acid residues that form at least oneof the complementary determining regions); more preferably, the bindingdomain is derived from a heavy chain camelid antibody, even morepreferably, the binding domain comprises a VHH sequence. Heavy chaincamelid antibodies, and the VHH-derived sequences are known to theperson skilled in the art. Camelid antibodies have been described,amongst others in WO9404678 and in WO2007118670, incorporated herein byreference. Still even more preferably, VHH comprises two disulphidebridges. Most VHH molecules have only one disulphide bridge; thepresence of an additional disulphide bridge will give extra stability tothe antibody domain, which is an advantageous characteristic for abinding domain that needs to be stable under harsh conditions. Mostpreferably, VHH preferably consists of a sequence selected from thegroup consisting of SEQ ID NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7, 3D10, 3D2,3D8, 3E6, 3F5, 3F7, 3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5,5D4, 5E5, 5F5, 5G2, 5G5, 5H5, 7A2, 7C2, 7D2, 7E1_1, 7F1, 8B10, 8B12,9A1, 9B5, 9C4, 9D5, 9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitablefragment thereof (which will then usually contain at least some of theamino acid residues that form at least one of the complementarydetermining regions) or homologues thereof. Homologues, as used here aresequences wherein each or any framework region and each or anycomplementary determining region shows at least 80% identity,preferably, at least 85% identity, more preferably, 90% identity, evenmore preferably, 95% identity with the corresponding region in thereference sequence (i.e., FR1_homologue versus FR1_reference,CDR1_homologue versus CDR1_reference, FR2_homologue versusFR2_reference, CDR2_homologue versus CDR2_reference, FR3_homologueversus FR3_reference, CDR3_homologue versus CDR3_reference andFR4_homologue versus FR4_reference) as measured in a BLASTp alignment(Altschul et al., 1997; FR and CDR definitions according to Kabat).

A second aspect hereof is a targeting agent, capable to retain anagrochemical on a plant and/or a plant part.

A “targeting agent,” as used herein, is a molecular structure,preferably with a polypeptide backbone, comprising at least one bindingdomain. A targeting agent in its simplest form consists solely of onesingle binding domain; however, a targeting agent can comprise more thanone binding domain and can be monovalent or multivalent and monospecificor multispecific, as further defined. Apart from one single or multiplebinding domains, a targeting agent can further comprise other moieties,which can be either chemically coupled or fused, whether N-terminally orC-terminally or even internally fused, to the binding domain. The othermoieties include, without limitation, one or more amino acids, includinglabeled amino acids (e.g., fluorescently or radio-actively labeled) ordetectable amino acids (e.g., detectable by an antibody), one or moremonosaccharides, one or more oligosaccharides, one or morepolysaccharides, one or more lipids, one or more fatty acids, one ormore small molecules or any combination of the foregoing. In oneembodiment, the other moieties function as spacers or linkers in thetargeting agent.

“Agrochemical,” as used herein, means any active substance or principlethat may be used in the agrochemical industry (including agriculture,horticulture, floriculture and home and garden uses, but also productsintended for non-crop related uses such as public health/pest controloperator uses to control undesirable insects and rodents, householduses, such as household fungicides and insecticides and agents, forprotecting plants or parts of plants, crops, bulbs, tubers, fruits(e.g., from harmful organisms, diseases or pests); for controlling,preferably promoting or increasing, the growth of plants; and/or forpromoting the yield of plants, crops or the parts of plants that areharvested (e.g., its fruits, flowers, seeds, etc.). Examples of suchsubstances will be clear to the skilled person and may, for example,include compounds that are active as insecticides (e.g., contactinsecticides or systemic insecticides, including insecticides forhousehold use), herbicides (e.g., contact herbicides or systemicherbicides, including herbicides for household use), fungicides (e.g.,contact fungicides or systemic fungicides, including fungicides forhousehold use), nematicides (e.g., contact nematicides or systemicnematicides, including nematicides for household use) and otherpesticides or biocides (for example, agents for killing insects orsnails); as well as fertilizers; growth regulators such as planthormones; micro-nutrients, safeners, pheromones; repellants; insectbaits; and/or active principles that are used to modulate (i.e.,increase, decrease, inhibit, enhance and/or trigger) gene expression(and/or other biological or biochemical processes) in or by the targetedplant (e.g., the plant to be protected or the plant to be controlled),such as nucleic acids (e.g., single-stranded or double-stranded RNA, as,for example, used in the context of RNAi technology) and other factors,proteins, chemicals, etc., known per se for this purpose, etc. Examplesof such agrochemicals will be clear to the skilled person; and, forexample, include, without limitation: glyphosate, paraquat, metolachlor,acetochlor, mesotrione, 2,4-D,atrazine, glufosinate, sulfosate,fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil,clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba,imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin,lambda-cyhalotrin, endosulfan, methamidophos, carbofuran, clothianidin,cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin,tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb,cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil,copper fungicides, trifloxystrobin, prothioconazole, difenoconazole,carbendazim, propiconazole, thiophanate, sulphur, boscalid and otherknown agrochemicals or any suitable combination(s) thereof. Othersuitable agrochemicals will be clear to the skilled person based on thedisclosure herein, and may, for example, be any commercially availableagrochemical, and, for example, include each of the compounds listed inPhillips McDougall, AgriService November 2007 V4.0, ProductsSection—2006 Market, Product Index pp. 10-20. The agrochemical can occurin different forms, including but not limited to, as crystals, asmicro-crystals, as nano-crystals, as co-crystals, as a dust, asgranules, as a powder, as tablets, as a gel, as a soluble concentrate,as an emulsion, as an emulsifiable concentrate, as a suspension, as asuspension concentrate, as a suspoemulsion, as a dispersion, as adispersion concentrate, as a microcapsule suspension or as any otherform or type of agrochemical formulation clear to those skilled in theart. Agrochemicals not only include active substances or principles thatare ready to use, but also precursors in an inactive form, which may beactivated by outside factors. As a non limiting example, the precursorcan be activated by pH changes, caused by plant wounds upon insectdamage, by enzymatic action caused by fungal attack, or by temperaturechanges or changes in humidity.

“Plant part,” as used herein, means any plant part whether part of anintact living plant or whether isolated or separated from an intactliving plant, and even dead plant material can be envisaged. Preferably,the plant parts are selected from the group consisting of leaves, stem,roots, fruits, cones, flowers, bulbs and tubers. More preferably, theplant parts are selected from the group consisting of leaves, stem androots. Even more preferably, the plant is an intact living plant and/orthe plant parts are plant parts of an intact living plant.

In order to be capable to retain an agrochemical on a plant or a plantpart, either one single or multiple targeting agents are either fusedwith or attached to the agrochemical, either by a covalent bond, byhydrogen bonds, by dipole-dipole interactions, by weak Van der Waalsforces or by any combination of the foregoing. “Attached,” as usedherein, means coupled to, connected to, anchored in, admixed with orcovering.

In one embodiment, the agrochemical is bound on or comprised in acarrier, as defined above, wherein the targeting agent is coupled eitherto the carrier or to the agrochemical. Preferably, the binding domain iscoupled to the carrier. “Coupled,” as used herein, can be any couplingallowing the retention of the agrochemical or carrier containing theagrochemical by the targeting agent; it can be a covalent as well as anon-covalent binding. Preferably, the coupling is a covalent binding. Itis clear to the person skilled in the art how binding domains and/ortargeting agents can be coupled to any type of functional groups presentat the outer surface of a carrier. “Functional group,” as used herein,means any chemical group to which a protein can be covalently bound,including but not limited to carboxyl-, amine-, hydroxyl-, sulfhydryl-,or alkynyl group. As a non-limiting example, coupling by forming of acarbodiimide bond between carboxyl groups on the outer surface of thecarrier and the amine-groups of the binding domain and/or targetingagent can be applied. Binding domains and/or targeting agents can becoupled with our without linking agents to the carrier. In the case of amicrobial cell or phage, the targeting agent hereof may be encoded bythe microbial cell or phage genome, whereas the agrochemical iscontained in or coupled to the microbial cell or phage, either as fusionprotein or by chemical linking. A “linking agent,” as used herein, maybe any linking agent known to the person skilled in the art; preferably,the linking agent is increasing the flexibility of the targeting agentbound on the carrier, thereby facilitating the binding of the bindingdomain comprised in the targeting agent to the binding site on theplant. Examples of such linking agents can be found in WO0024884 andWO0140310.

The carrier may be a microcarrier. A “microcarrier,” as used herein, isa particulate carrier where the particles are less than 500 μm indiameter, preferably, less than 250 μm, even more preferably, less than100 μm, most preferably, less than 50 Microcarriers for delivery ofagrochemicals are known to the person skilled in the art, and include,but are not limited to nanocapsules, microcapsules, nanospheres,microspheres, weak ionic resin particles, polymer particles, compositegel particles, particles made from artificially lignified cellulose,liposomes, vesicles and cochleate delivery vehicles. It is also possiblethat one or more agrochemicals are either present on or within amicrobial cell (e.g., a yeast cell) or a phage (for example, because theone or more agrochemicals can be loaded into (or onto) such cells or arebiologicals that have been produced/expressed in the microbial cell) orthat the one or more agrochemicals are associated (e.g., bound to orembedded in) with cell fragments (e.g., fragments of cells walls or cellmembranes), cell fractions or other cell debris (for example, obtainedby fractionating or lysing the microbial cells into (or onto) which theone or more agrochemicals have been loaded, produced or expressed) andthat therefore the microbial cells or phages are used as microcarriers.As used herein microcarrier, microparticle, microsphere, microcapsule,nanoparticle, nanocapsule and nanosphere can be used interchangeably.Such microcarriers have been described, amongst others, in U.S. Pat. No.6,180,141, WO2004004453, WO2005102045 and U.S. Pat. No. 7,494,526,incorporated here by reference. Preferably, the microcarrier is amicroparticle composed of a natural polymer. Characteristics ofmicrocarriers can be such that they enable slow release of theagrochemical, delayed release of the agrochemical or immediate releaseof the agrochemical, all types of microcarriers have their specific use.Microcarriers may naturally comprise cross-linkable residues suitablefor covalent attachment or microcarriers may be derivatized to introducesuitable cross-linkable groups to methods well known in the art. Suchderivatization may occur prior to manufacturing of the microcarrier,i.e., at the level of the raw materials that will be used in themanufacturing process, it may occur during the manufacturing process ofthe microcarrier or it may occur subsequent to the manufacturing of themicrocarrier. In one specific embodiment, functional groups on themicrocarrier may be bound to a linking agent or spacer, which is on itsturn bound to a targeting agent as defined above.

In another embodiment, one or more binding domains comprised in thetargeting agent, bind to a binding site or to an antigen comprised insuch binding site, present in or on one or more particular parts of theplant, preferably the intact living plant. Preferably, the parts of theplant, more preferably of the intact living plant, are selected from thegroup consisting of leaves, stem, roots, fruits, cones, flowers, bulbsor tubers. More preferably, the parts of the plant, preferably theintact living plant, are selected from the group consisting of leaves,stem or roots. More preferably, one or more binding domains comprised inthe targeting agent, bind to a binding site or to an antigen comprisedin such binding site, on the surface of the plant, preferably the intactliving plant. Preferably, the surface of the plant, preferably theintact living plant, is the surface of a part of the plant, preferablythe intact living plant, selected from the group consisting of leafsurface, stem surface, root surface, fruit surface, cone surface, flowersurface, bulb surface or tuber surface; even more preferably, thesurface of the plant, preferably the intact living plant, is the surfaceof a part of the plant, preferably the intact living plant, selectedfrom the group consisting of root surface, stem surface and leafsurface.

In another embodiment, one or more binding domains comprised in thetargeting agent, bind to binding site, or to an antigen comprised insuch binding site, in or on a particular structure of the plant,preferably the intact living plant, or in or on a particular structureof a particular part of the plant, preferably the intact living plant;more preferably, in or on a particular structure involved or implicatedto be involved in transport of nutrients, agrochemicals or otherchemicals into the plant and/or involved or implicated to be involved inplant defense. Preferably, the particular structure is selected from thegroup consisting of trichomes, stomata, lenticels, thorns, spines, roothairs, cuticle and wax layer, even more preferably, the particularstructure is selected from the group consisting of trichomes, stomataand cuticle. In one embodiment, the one or more binding domainscomprised in the targeting agent, bind to binding site, or to an antigencomprised in such binding site, in or on plant trichomes. In anotherembodiment, the one or more binding domains comprised in the targetingagent, bind to binding site, or to an antigen comprised in such bindingsite, in or on stomata. In yet another embodiment, the one or morebinding domains comprised in the targeting agent, bind to binding site,or to an antigen comprised in such binding site, in or on plant cuticle.

In yet another embodiment, one or more binding domains hereof andcomprised in the targeting agent, bind to gum arabic. In anotherembodiment, one or more of the binding domains comprised in thetargeting agent, bind to lectins, lectin-like domains, extensins, orextensin-like domains; more preferably, the binding domain is bindingpotato lectin. Preferably, one or more of the binding domains comprisedin the targeting agent comprises four framework regions and threecomplementary determining regions, or any suitable fragment thereof(which will then usually contain at least some of the amino acidresidues that form at least one of the complementary determiningregions); more preferably, one or more of the binding domains comprisedin the targeting agent is derived from a heavy chain camelid antibody,even more preferably, one or more of the binding domains comprised inthe targeting agent comprises a VHH sequence. Still even morepreferably, VHH comprises two disulphide bridges. Most preferably, VHHpreferably consists of a sequence selected from the group consisting ofSEQ ID NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7, 3D10, 3D2, 3D8, 3E6, 3F5, 3F7,3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5, 5D4, 5E5, 5F5, 5G2,5G5, 5H5, 7A2, 7C2, 7D2, 7E1_1, 7F1, 8B10, 8B12, 9A1, 9B5, 9C4, 9D5,9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitable fragment thereof(which will then usually contain at least some of the amino acidresidues that form at least one of the complementary determiningregions) or homologues thereof.

A third aspect hereof is the use of a targeting agent hereof to deliverand retain an agrochemical or a combination of agrochemicals to a plantor plant part.

Any plant part whether part of an intact living plant or whetherisolated or separated from an intact living plant, and even dead plantmaterial can be envisaged as a target to deliver and retain anagrochemical or a combination of agrochemicals using a targeting agenthereof. Preferably, the plant parts are selected from the groupconsisting of leaves, stem, roots, fruits, cones, flowers, bulbs andtubers. More preferably, the plant parts are selected from the groupconsisting of leaves, stem and roots. Even more preferably, the plant isan intact living plant and/or the plant parts are plant parts of anintact living plant. Delivery is carried out using any suitable ordesired manual or mechanical technique for application of anagrochemical or a combination of agrochemicals, including but notlimited to spraying, brushing, dressing, dripping, coating, dipping,spreading, applying as small droplets, a mist or an aerosol. Asnon-limiting examples, a targeting agent hereof can be used to deliverand retain an agrochemical or a combination of agrochemicals to thefoliage of a field grown crop, it can be used to deliver and retain anagrochemical or a combination of agrochemicals to the roots of a croppropagated by hydroculture, it can be used to deliver and retain anagrochemical or a combination of agrochemicals to harvested plant parts(e.g., fruits, flowers or seeds) as a post-harvest treatment, it can beused to deliver and retain an agrochemical or a combination ofagrochemicals to living or dead plant material present in the soil uponpreparation of arable land, which is particularly useful in combinationwith no tilling agricultural practices, or it can be used to deliver andretain an agrochemical to a substrate placed in the vicinity of arhizosphere to achieve distribution and prolonged retention ofagrochemicals throughout the rhizosphere. One particularly advantageousaspect hereof is that it allows, by suitably choosing the combination oftargeting agent and agrochemical, or combination of agrochemicals toformulate the same active substance for a variety of different uses, forexample, on different plant species or parts of plants, for differentenvironmental conditions (type of soil, amount of rainfall and otherweather conditions, or even different seasonal conditions) and differentend-uses (for example, in the field, in greenhouses, in gardens, inhydroponic culture systems, for possibly environmental dependent quick,delayed or slow release use, for household use and for use by pestcontrol operators). Thus, by the use of the targeting agent to deliverand retain the agrochemical, it is possible, starting from activeagrochemical substances or agrochemical formulations with provenefficacy, that are environmentally acceptable, to provide a range ofdifferent and improved plant protection products or agents or otheragrochemical products that are tailored for desired or intended end use.As a non-limiting example, a broad spectrum herbicide can be made plantspecies specific by delivering it using a targeting agent comprising aplant species specific binding domain; on the other hand, delivering thesame herbicide using a targeting agent comprising a binding domain thathas a broad spectrum specificity can help to reduce the amounts ofherbicide needed to exert its desired action. Also, undesired off-targetactivity of an agrochemical, e.g., versus beneficial insects, can beavoided by delivering the agrochemical using a targeting agentcomprising a binding domain that is highly specific for the targetedcrop or for specific parts of the targeted crop.

Preferably, the agrochemical or combination of agrochemicals is selectedfrom the groups consisting of herbicides, insecticides, fungicides,nematicides, biocides, fertilizers, safeners, micro-nutrients and plantgrowth regulating compounds.

Preferably, the method of delivery and retention of an agrochemical orcombination of agrochemicals results in improved deposition of theagrochemical or combination of agrochemicals on the plant or plant part.“Improved deposition,” as used herein, means that either the quantity ofthe agrochemical or combination of agrochemicals that is bound to theplant or plant part is increased and/or that the distribution of theagrochemical or combination of agrochemicals is divided over the plantor plant part either more equally or more concentrated in function ofthe specificity of the binding domain comprised in the targeting agent,when compared to the same agrochemical or combination of agrochemicalsapplied without the use of any targeting agent.

In one embodiment, the agrochemical or combination of agrochemicals isbound on or comprised in a carrier, preferably, a microcarrier asdefined earlier. This may, for example, be particularly advantageous foran agrochemical or combination of agrochemicals that are volatile orrapidly degradable by environmental factors such as the presence ofmoisture or UV-irradiation, or that pose a considerable toxicity hazardfor the person handling the agrochemical or combination ofagrochemicals. In one specific embodiment, functional groups on thecarrier may be bound to a linking agent or spacer, which is on its turnbound to a targeting agent as defined above.

A fourth aspect hereof is a composition, comprising at least (i) onetargeting agent comprising at least one binding domain hereof and (ii)an agrochemical or combination of agrochemicals.

The targeting agent(s) comprised in the composition may either be a“mono-specific” targeting agent or a “multi-specific” targeting agent.By a “mono-specific” targeting agent is meant a targeting agent thatcomprises either a single binding domain, or that comprises two or moredifferent binding domains that each are directed against the sameantigen present at or in the same binding site or that form the bindingsite. Thus, a mono-specific targeting agent is capable of binding to asingle binding site, either through a single binding domain or throughmultiple binding domains. By a “multi-specific” targeting agent is meanta targeting agent that comprises two or more binding domains that areeach directed against different antigens present at or in a binding siteor that form the binding site. Thus, a “bi-specific” targeting agent iscapable of binding to two different binding sites or antigens present ator in a binding site or that form the binding site; a “tri-specific”targeting agent is capable of binding to three different antigenspresent at or in a binding site or that form the binding site; and so onfor “multi-specific” targeting agents. Also, in respect of the targetingagents described herein, the term “monovalent” is used to indicate thatthe targeting agent comprises a single binding domain; the term“bivalent” is used to indicate that the targeting agent comprises atotal of two single binding domains; the term “trivalent” is used toindicate that the targeting agent comprises a total of three singlebinding domains; and so on for “multivalent” targeting agents.Accordingly, in one aspect, the above composition hereof comprises twoor more identical or different targeting agents, by which is meant twoor more targeting agents that, for identical targeting agents, each bindto identical or different antigens present at or in the same bindingsite, whereas for different targeting agents, at least one binds todifferent antigens present at or in the same binding site or indifferent binding sites.

Preferably, the targeting agent(s) comprised in the composition,comprise at least one binding domain that binds to a binding site or toan antigen comprised in such binding site, present in or on one or moreparticular parts of a plant, preferably of an intact living plant.Preferably, the parts of the plant, more preferably, of the intactliving plant, are selected from the group consisting of leaves, stems,roots, fruits, cones, flowers, bulbs or tubers. More preferably, theparts of the intact living plant are selected from the group consistingof leaves, stems or roots. More preferably, the targeting agent(s)comprised in the composition, comprise at least one binding domain thatbinds to a binding site or to an antigen comprised in such binding site,on the surface of the intact living plant. Preferably, the surface ofthe intact living plant is the surface of a part of the intact livingplant, selected from the group consisting of leaf surface, stem surface,root surface, fruit surface, cone surface, flower surface, bulb surfaceor tuber surface; even more preferably, the surface of the intact livingplant is the surface of a part of the intact living plant, selected fromthe group consisting of root surface, stem surface and leaf surface.

Preferably, the targeting agent(s) comprised in the composition,comprise at least one binding domain that binds to a binding site, or toan antigen comprised in such binding site, in or on a particularstructure of the plant, preferably the intact living plant or in or on aparticular structure of a particular part of the plant, preferably theintact living plant; more preferably, in or on a particular structureinvolved or implicated to be involved in transport of nutrients,agrochemicals or other chemicals into the plant and/or involved orimplicated to be involved in plant defense. Preferably, the particularstructure is selected from the group consisting of trichomes, stomata,lenticels, thorns, spines, root hairs, cuticle and wax layer, even morepreferably, the particular structure is selected from the groupconsisting of trichomes, stomata and cuticle. In one embodiment, thetargeting agent(s) comprised in the composition, comprise at least onebinding domain that binds to a binding site, or to an antigen comprisedin such binding site, in or on plant trichomes. In another embodiment,the targeting agent(s) comprised in the composition, comprise at leastone binding domain that binds to a binding site, or to an antigencomprised in such binding site, in or on stomata. In yet anotherembodiment, the targeting agent(s) comprised in the composition,comprise at least one binding domain that binds to a binding site, or toan antigen comprised in such binding site, in or on plant cuticle.

In yet another embodiment, the targeting agent(s) comprised in thecomposition, comprise at least one binding domain that binds to gumarabic. In preferred embodiment, the targeting agent(s) comprised in thecomposition, comprise at least one binding domain that binds to lectins,lectin-like domains, extensins, or extensin-like domains; morepreferably, the binding domain is binding potato lectin. Preferably, thetargeting agent(s) comprised in the composition, comprise at least onebinding domain that comprises four framework regions and threecomplementary determining regions, or any suitable fragment thereof(which will then usually contain at least some of the amino acidresidues that form at least one of the complementary determiningregions); more preferably, one or more of the binding domains comprisedin the targeting agent is derived from a heavy chain camelid antibody,even more preferably, one or more of the binding domains comprised inthe targeting agent comprises a VHH sequence. Still even morepreferably, VHH comprises two disulphide bridges. Most preferably, VHHcomprises, preferably, consists of a sequence selected from the groupconsisting of SEQ ID NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7, 3D10, 3D2, 3D8,3E6, 3F5, 3F7, 3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5, 5D4,5E5, 5F5, 5G2, 5G5, 5H5, 7A2, 7C2, 7D2, 7E1_1, 7F1, 8B10, 8B12, 9A1,9B5, 9C4, 9D5, 9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitablefragment thereof (which will then usually contain at least some of theamino acid residues that form at least one of the complementarydetermining regions) or homologues thereof.

In the composition hereof, the agrochemical or combination ofagrochemicals are preferably selected from the group consisting ofherbicides, insecticides, fungicides, nematicides, biocides,fertilizers, safeners, micro-nutrients or plant growth regulatingcompounds.

In the composition hereof, the agrochemical or combination ofagrochemicals may be in a liquid, semi-solid or solid form and, forexample, be maintained as an aerosol, flowable powder, wettable powder,wettable granule, emulsifiable concentrate, suspension concentrate,microemulsion, capsule suspension, dry microcapsule, tablet or gel or besuspended, dispersed, emulsified or otherwise brought in a suitableliquid medium (such as water or another suitable aqueous, organic oroily medium) for storage or application onto a plant. Optionally, thecomposition further comprises one or more further components such as,but not limited to diluents, solvents, adjuvants, surfactants, wettingagents, spreading agents, oils, stickers, thickeners, penetrants,buffering agents, acidifiers, anti-settling agents, anti-freeze agents,photo-protectors, defoaming agents, biocides and/or drift control agentsor the like, suitable for use in the composition hereof.

In one embodiment, the agrochemical or combination of agrochemicals isbound on or otherwise comprised in a carrier. In the case of acombination of agrochemicals, each individual agrochemical may be boundon or otherwise comprised in an individual carrier, or a suitablecombination of agrochemicals may be jointly bound on or otherwisecomprised in one carrier. As an alternative to the use of a carrier, theagrochemical or combination of agrochemicals may also be provided in theform of (small) particles which are provided with a suitable coating or(outside) layer to which the targeting agent is coupled or can bind andwhich may also serve to stabilize or improve the physical integrity orstability of the particles. As another alternative, the agrochemical orcombination of agrochemicals may be suitably mixed with an excipient orbinder to which the targeting agent is coupled or can bind, and whichmay again also serve to stabilize or improve the physical integrity orstability of the particles. Such coated or composite particles arepreferably in the form of a slurry, wet cake or free-flowable powder,tablet, capsule or liquid concentrate (such as an emulsion, suspensionor dispersion).

In one embodiment, the composition hereof is for agrochemical use.“Agrochemical use,” as used herein, not only includes the use ofagrochemicals as defined above (for example, pesticides, growthregulators, nutrients/fertilizers, repellants, defoliants, etc.) thatare suitable and/or intended for use in field grown crops (e.g.,agriculture), but also includes the use of agrochemicals as definedabove (for example, pesticides, growth regulators,nutrients/fertilizers, repellants, defoliants, etc.) that are meant foruse in greenhouse grown crops (e.g., horticulture/floriculture) orhydroponic culture systems and even the use of agrochemicals as definedabove that are suitable and/or intended for non-crop uses such as usesin private gardens, household uses (for example, herbicides orinsecticides for household use), or uses by pest control operators (forexample, weed control, etc.).

Based on the teaching set out in the present specification, and, forexample, depending on the agrochemical(s) to be delivered, on thepart(s) to the plant to which the agrochemical(s) is to be delivered,and the intended agrochemical action of the composition hereof (and/orthe agrochemical(s) included therein), the skilled person will be ableto suitably select the specific binding domains/targeting agent thatcan/should be present in the composition hereof (as well as the othercomponents of the composition, such as the carrier, the agrochemical andthe agrochemical form/formulation) in order to achieve thedesired/intended agrochemical action. Thus, with advantage, based on thedisclosure herein, it is possible for the skilled person to suitablyselect a suitable combination of binding domain(s)/targeting agent(s),agrochemical(s), carrier, further components of the composition and theagrochemical form/formulation of the composition in order to achieve theintended/desired agrochemical action. In this respect, it should benoted that, as currently contemplated, and although it is foreseen thatsome such combinations will be more efficacious and/or more preferredthan others, there will likely be multiple such combinations possiblethat will give the intended/desired agrochemical action to the more orless same degree. This also allows the skilled person to take intoaccount other (secondary) factors when selecting the combination to beused, such as the specific crop(s) to be protected, the prevalent field,soil, weather and/or other environmental conditions, the way thatcomposition is preferably applied, the environment in which it isapplied (field, greenhouse, etc.), the desired persistence and/or otherfactors that may influence the choice of an agrochemical composition fora specific application.

For example, and without limitation, when the composition hereof isintended to bind to one or more specific parts of the plant, thetargeting agent (i.e., the one or more binding domains present therein)are preferably directed towards one or more binding sites (as definedherein) that are present (i.e., in a sufficient amount) in/on thepart(s) of the plant (it also being possible that such binding site(s)are present in/on the part(s) of the plant in a larger amount(s)/to agreater degree than on other part(s) of the plant, i.e., so as toprovide a binding domain/targeting agent/composition hereof that canpreferentially bind to the intended/desired part(s) of the plantcompared to one or more other parts of the plant); and compositionshereof that comprise such binding domains/targeting agents (i.e., suchthat the compositions are directed towards binding sites present in thedesired part(s) of the plant and, preferably, such that they can bindpreferentially to the desired part(s) of the plant) form some specificbut non-limiting aspects hereof. For example, and without limitation:

-   -   for a composition hereof that is intended to bind to the leaves        of a plant, the binding domains and/or targeting agent may be        directed against one or more of the following binding sites on        (the leaves of) a plant: cutin, cuticular waxes,        arabinogalactan-proteins or lipid transfer proteins;    -   for a composition hereof that is intended to bind to the roots        of a plant, the binding domains and/or targeting agent may be        directed against one or more of the following binding sites on        (the roots of) a plant: extensins or pectins;    -   for a composition hereof that is intended to bind to the stem of        a plant, the binding domains and/or targeting agent may be        directed against one or more of the following binding sites on        (the stem of) a plant: lignins, extensins or excretion products;        and each such composition hereof forms a specific, but        non-limiting aspect hereof.

A fifth aspect hereof is a composition, comprising at least (i) onetargeting agent comprising at least one binding domain hereof and (ii) acarrier.

The targeting agent(s) comprised in the composition may either bemono-specific targeting agents or multi-specific targeting agents andmay be either monovalent targeting agents or multivalent targetingagents. Accordingly, in one aspect, the above composition hereofcomprises two or more identical or different targeting agents, by whichis meant two or more targeting agents that, for identical targetingagents, each bind to identical or different antigens present at or inthe same binding site, whereas for different targeting agents, at leastone binds to different antigens present at or in the same binding siteor in different binding sites.

In one specific embodiment, which is preferred but non-limiting, thecarrier is such that it allows the composition hereof to be suitablyapplied to the intended site of action, and/or such that it allows thecomposition hereof to be formulated such that it can be suitably appliedto the intended site of action; using any suitable or desired manual ormechanical technique such as spraying, brushing, dripping, dipping,coating, spreading, applying as small droplets, a mist or an aerosol,etc. Examples of such techniques, of compositions hereof that aresuitable for use in such techniques, and of methods for making andformulating such compositions hereof will be clear to the skilled personbased on the disclosure herein. Preferably, the carrier is such that oneor more active substances can be incorporated, encapsulated or includedinto the carrier, e.g., as a nanocapsule, microcapsule, nanosphere,micro-sphere, liposome or vesicle. Even more preferably, the carrier issuch that upon such incorporation, encapsulation, embedding orinclusion, the complex thus obtained can be suspended, dispersed,emulsified or otherwise brought into a suitable liquid medium (such aswater or another suitable aqueous, organic or oily medium) so as toprovide a (concentrated) liquid composition hereof that has a stabilitythat allows the composition hereof to be suitably stored or (wherenecessary after further dilution) applied to the intended site ofaction. Even more preferably, the carrier is such that the compositionhereof can be transported and/or stored prior to final use, optionally(and usually preferably) as a suitable liquid concentrate, dry powder,tablet, capsule, slurry or “wet cake,” which can be suitably diluted,dispersed, suspended, emulsified or otherwise suitably reconstituted bythe end user prior to final use (and such concentrates form a furtheraspect hereof). Carriers, preferably microcarriers, suitable for thispurpose (and methods for absorbing, encapsulating, embedding, etc., theactive principles therein) will be clear to the skilled person based onthe disclosure herein and/or may be commercially available. Somenon-limiting examples include solid or semi-solid microspheres orgranulates in which the active ingredients are embedded or absorbed in asuitable matrix material or microcapsules comprising a shell materialthat surround a core that contains the active ingredient (i.e.,encapsulated within the microcapsule).

Preferably, the carriers are such that they have immediate, delayed,gradual, triggered or slow release characteristics, for example, overseveral minutes, several hours, several days or several weeks. Also, thecarriers may be made of materials (e.g., polymers) that rupture orslowly degrade (for example, due to prolonged exposure to high or lowtemperature, high or low pH, sunlight, high or low humidity or otherenvironmental factors or conditions) over time (e.g., over minutes,hours, days or weeks) or that rupture or degrade when triggered byparticular external factors (such as high or low temperature, high orlow pH, high or low humidity or other environmental factors orconditions) and so release the active agent from the microcapsule. Thecarrier is also, preferably, such that the agrochemicals are releasedfrom the carrier when the composition hereof is applied to the intendedsite of action, i.e., at a rate that is sufficient to provide thedesired action of the agrochemicals during the desired period of time(e.g., the time between two applications of the composition hereof).

In one particular embodiment, the carrier, preferably the microcarrier,may be composed of polymer materials, such as, for example,poly-urethane, poly-urea, poly-amide, poly-ethylene,polyethylene-glycol, polyvinyl alcohols, melamine, urea/formaldehyde,acrylic polymers, nylon, vinyl acetate or siloxane polymersor—optionally (and usually preferably) for agrochemicalpurposes—biodegradable polymers (such as, for example, agar, gelatin,alginates, gums, pectins, poly-alcohols such as cetyl-alcohol, oilysubstances such as hydrogenated palm oil or soybean oil, starches,waxes, etc. Alternatively, and although this is usually less preferred,non-biodegradable materials may be used such as poly-methylacrylates,poly-ethersulfones, metal oxides, carbon structures, etc.

Preferably, the carrier is selected from the group consisting ofnanocapsules, nanospheres, microcapsules, microspheres, polymerparticles, particles made from artificially lignified cellulose,composite gel particles, weak ionic resin particles, microbial cells orfragments thereof. More preferably, the carrier is selected from thegroup consisting of microcapsules, microspheres or polymer particles.Most preferably, the carrier is a microcapsule.

In one embodiment, the targeting agent(s) comprised in the composition,comprise at least one binding domain that comprises four frameworkregions and three complementary determining regions, or any suitablefragment thereof (which will then usually contain at least some of theamino acid residues that form at least one of the complementarydetermining regions); more preferably, one or more of the bindingdomains comprised in the targeting agent is derived from a heavy chaincamelid antibody, even more preferably, one or more of the bindingdomains comprised in the targeting agent comprises a VHH sequence. Stilleven more preferably, VHH comprises two disulphide bridges. Mostpreferably, VHH comprises, preferably, consists of a sequence selectedfrom the group consisting of SEQ ID NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7,3D10, 3D2, 3D8, 3E6, 3F5, 3F7, 3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6,5C4, 5C5, 5D4, 5E5, 5F5, 5G2, 5G5, 5H5, 7A2, 7C2, 7D2, 7E1_1, 7F1, 8B10,8B12, 9A1, 9B5, 9C4, 9D5, 9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or anysuitable fragment thereof (which will then usually contain at least someof the amino acid residues that form at least one of the complementarydetermining regions) or homologues thereof.

In another embodiment, the targeting agent and the carrier comprised inthe composition hereof are coupled to each other. Preferably, the onesingle targeting agent or multiple targeting agents are coupled to thecarrier by affinity binding or by covalent binding. More preferably, theone single targeting agent or multiple targeting agents, are coupled tothe carrier by covalent binding. Preferably, the one single targetingagent or multiple targeting agents are coupled, preferably covalentlycoupled, to the carrier by the use of a functional group present on theouter surface of the carrier. Preferably, the binding domain comprisedin the targeting agent(s) is coupled, preferably covalently coupled, tothe carrier. Alternatively, the one single targeting agent or multipletargeting agents are coupled, preferably covalently coupled, to thecarrier via a moiety that is not the binding domain comprised in thetargeting agent.

In yet another embodiment, the carrier is coupled to and/or comprises atleast one agrochemical as defined above. Preferably, the agrochemical isselected from the group consisting of herbicides, insecticides,fungicides, nematicides, biocides, fertilizers, micro-nutrients,safeners or plant growth regulating compounds. In this embodiment, thecomposition is for agrochemical use.

The carrier with the one or more targeting agents bound, coupled orotherwise attached thereto or associated therewith may be dissolved,emulsified, suspended or dispersed or otherwise included into a suitableliquid medium (such as water or another aqueous, organic or oily medium)so as to provide a (concentrated) solution, suspension, dispersion oremulsion that is suitable for storage.

For example, when the composition hereof is intended for agrochemicaluse, the composition hereof may be in a liquid, semi-solid or solid formthat is suitable for spraying, such as a solution, emulsion, suspension,dispersion, aerosol, flowable powder or any other suitable form.

In particular, such a composition hereof for agrochemical use maycomprise a microcapsule, microsphere, nanocapsule, nanosphere, liposomesor vesicles, etc., in which the one or more agrochemicals are suitablyencapsulated, enclosed, embedded, incorporated or otherwise included;and one or more targeting agents that each comprise one or more bindingdomains for binding to one or more antigens present at or in the bindingsite or that form the one or more binding sites on a plant or parts of aplant, such as a leaf, stem, flower, fruit, bulb or tuber of a plant).

A sixth aspect hereof is a method for delivery of an agrochemical or acombination of agrochemicals to a plant, the method comprising at leastone application of a composition hereof to the plant.

“One application,” as used herein, means a single treatment of a plantor plant part. According to this method, either the composition hereofis applied as such to the plant or plant part, or the composition isfirst dissolved, suspended and/or diluted in a suitable solution beforebeing applied to the plant. The application to the plant is carried outusing any suitable or desired manual or mechanical technique forapplication of an agrochemical or a combination of agrochemicals,including but not limited to spraying, brushing, dressing, dripping,dipping, coating, spreading, applying as small droplets, a mist or anaerosol. Upon such application to a plant or part of a plant, thecomposition can bind at or to the binding site (or to one or moreantigens present at or in the binding site or that form the bindingsite) via one or more binding domains that form part of the targetingagent(s) comprised in the composition, preferably in a targeted manner.Thereupon, the agrochemicals are released from the carrier (e.g., due todegradation of the carrier or passive transport through the wall of thecarrier) in such a way that they can provide the desired agrochemicalaction(s). A particular advantage of delivering an agrochemical orcombination of agrochemicals to a plant using a composition hereof isthat it may lead to an improved deposition (as defined earlier) of theagrochemical or combination of agrochemicals on the plant or plant partand/or an increased resistance of the agrochemical or combination ofagrochemicals against loss due to external factors such as rain,irrigation, snow, hail or wind.

In one embodiment, delivering an agrochemical or combination ofagrochemicals to a plant using a composition hereof results in improvedrainfastness of the agrochemical or combination of agrochemicals.“Improved rainfastness,” as used herein, means that the percentage lossof agrochemical or combination of agrochemicals, calculated before andafter rain, is smaller when the agrochemical or combination ofagrochemicals is applied in a composition hereof, compared with the sameagrochemical or combination of agrochemicals comprised in a comparablecomposition, without any targeting agent. A “comparable composition,” asused herein, means that the composition is identical to the compositionhereof, apart from the absence of the targeting agent used in thecomposition hereof.

In one embodiment, a suitable dose of the agrochemical or combination ofagrochemicals comprised in a composition hereof is applied to the plantor plant part. A “suitable dose,” as used herein, means an efficaciousamount of active substance of the agrochemical comprised in thecomposition.

Preferably, the method comprises the application of a meaningfullyreduced dose of an agrochemical or combination of agrochemicals to theplant, to obtain similar beneficial effects for the agrochemical orcombination of the agrochemicals, as compared with the application ofthe same agrochemical or combination of agrochemicals comprised in acomparable composition, as defined above, without any targeting agent.The meaningful reduction is obtained by directing the agrochemical tothe plant using targeting agents hereof. Alternatively, the methodcomprises an application of a suitable dose, wherein the applicationfrequency is meaningfully reduced, to obtain similar beneficial effectsfor the agrochemical, compared with the frequency of application of thesame dose of an encapsulated composition of the agrochemical lacking thepresence of a targeting agent hereof. Even more preferably, the methodcomprises an application wherein the suitable dose as well as theapplication frequency are both significantly reduced to obtain similarbeneficial effects for the agrochemical, compared with the suitable doseand application frequency of a an encapsulated composition of theagrochemical lacking the presence of a targeting agent hereof.

A seventh aspect hereof is a method for protecting a plant againstexternal (biotic or abiotic) stress and/or to modulate the viability,growth or yield of a plant or plant parts and/or to modulate geneexpression in a plant or plant part resulting in alteration of (levelsof) plant constituents (such as proteins, oils, carbohydrates,metabolites, etc.), the method comprising at least one application of acomposition hereof. If needed, the composition is dissolved, suspendedand/or diluted in a suitable solution. “Protecting a plant,” as usedhere, is the protection of the plant against any stress; the stress maybe biotic stress, such as, but not limited to, stress caused by weeds,insects, rodents, nematodes, mites, fungi, viruses or bacteria, or itmay be abiotic stress, such as but not limited to drought stress, saltstress, temperature stress or oxidative stress.

Preferably, the method comprises the application of a meaningfullyreduced dose of an agrochemical or combination of agrochemicals to theplant, to obtain similar beneficial effects for the agrochemical orcombination of the agrochemicals, as compared with the application ofthe same agrochemical or combination of agrochemicals comprised in acomparable composition, as defined earlier, without any targeting agent.The meaningful reduction is obtained by directing the agrochemical tothe plant using targeting agents hereof. Alternatively, the methodcomprises an application of a suitable dose, wherein the applicationfrequency is meaningfully reduced, to obtain similar beneficial effectsfor the agrochemical, compared with the frequency of application of thesame dose of an encapsulated composition of the agrochemical lacking thepresence of a targeting agent hereof. Even more preferably, the methodcomprises an application wherein the suitable dose as well as theapplication frequency are both significantly reduced to obtain similarbeneficial effects for the agrochemical, compared with the suitable doseand application frequency of an encapsulated agrochemical lacking thepresence of a targeting agent hereof.

An eighth aspect hereof is a method for manufacturing a specificallytargeting agrochemical carrier, the method comprising (a) packing anagrochemical in or on(to) a carrier and (b) attaching at least onetargeting agent hereof to the carrier.

“Packing,” as used herein, means incorporating, including, immobilizing,adsorbing, absorbing, binding, encapsulating, embedding, attaching,admixing, anchoring or comprising. Methods for packing an agrochemical,as defined above, in or on(to) a carrier are known to the person skilledin the art and include, without limitation, drip-casting, extrusiongranulation, fluid bed granulation, co-extrusion, spray drying, spraychilling, atomization, addition or condensation polymerization,interfacial polymerization, in situ polymerization, coacervation, sprayencapsulation, cooling melted dispersions, solvent evaporation, phaseseparation, solvent extraction, sol-gel polymerization, high or lowshear mixing, fluid bed coating, pan coating, melting, passive or activeabsorption or adsorption. In one preferred, but not limiting,embodiment, an agrochemical is packed into a microcarrier using suitablemicroencapsulation techniques, such as interfacial polymerization, insitu polymerization, coacervation, spray encapsulation, cooling melteddispersions, solvent evaporation, phase separation, solvent extractionor sol-gel polymerization. Preferred, but non-limiting examples ofsuitable materials for producing such microcarriers are materials suchas alginates, agar, gelatin, pectins, gums, hydrogenated oils, starches,waxes, polyalcohols, poly-urea, poly-urethane, poly-amide, melamine,urea/formaldehyde, nylon and other (optionally and usually preferredbiodegradable or inert) polymers. More preferably, at least onefunctional group is present at the outer surface of the microcarrier.

At least one targeting agent hereof is attached to the carrier, eitherby a covalent bond, by hydrogen bonds, by dipole-dipole interactions, byweak Van der Waals forces or by a combination of any of the foregoing.Attachment of the targeting agent to the carrier may be performed whilepacking the agrochemical in or on(to) the carrier, it may be performedsubsequent to packing of the agrochemical in or on(to) the carrier or itmay be performed only at the time the agrochemical containing carrier isdissolved in a suitable solution for application. Suitable processes forattaching the targeting agent to a carrier will be clear to the personskilled in the art. In one embodiment, the targeting agent and thecarrier are coupled to each other. Preferably, the targeting agent(s)are coupled to the carrier by affinity binding or by covalent binding.More preferably, the targeting agent(s) are coupled to the carrier bycovalent binding. Preferably, the targeting agent(s) are coupled,preferably covalently coupled, to the carrier by the use of a functionalgroup present on the outer surface of the carrier. Preferably, thebinding domain comprised in the targeting agent(s) is coupled,preferably covalently coupled, to the carrier. Alternatively, thetargeting agent(s) are coupled, preferably covalently coupled, to thecarrier via a moiety that is not the binding domain comprised in thetargeting agent. In one embodiment, the process for attaching thetargeting agent(s) to a carrier comprises (a) reacting a linking agentwith a carrier, and (b) reacting at least one targeting agent with thelinking agent.

A ninth aspect hereof is a process for attaching a targeting agenthereof to a carrier, comprising (a) reacting a linking agent with acarrier, and (b) reacting the targeting agent with the linking agent.“Reacting,” as used herein, means that the linking agent is placed inconditions allowing the binding of the linking agent to the carrierand/or the targeting agent.

A tenth aspect hereof is a specifically targeting agrochemical carrier,obtained by the above described method. “Specifically targeting,” asused herein, means that the carrier can bind specifically to a bindingsite on a plant or on a plant part, through at least one targeting agenthereof, which is attached, preferably coupled, most preferablycovalently bound, to the carrier.

A last aspect hereof is the use of any binding domain hereof to isolateamino acid sequences that are responsible for specific binding to thebinding site or to an antigen comprised in the binding site and toconstruct artificial binding domains based on the amino acid sequences.Indeed, in the binding domains hereof, the framework regions and thecomplementary determining regions are known, and the study ofderivatives of the binding domain, binding to the same binding site orantigen comprised in the binding site, will allow deducing the essentialamino acids involved in binding the binding site or antigen comprised inthe binding site. This knowledge can be used to construct a minimalbinding domain and to create derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G: Binding domains (VHH) binding to leaf surface.

FIG. 1A: VHH3E6 5 μg/ml in PBS binding to native potato leaf surface.Detection with anti-histidine antibodies directly conjugated withAlexa-488 fluorescent dye. VHH 3E6 is binding leaf surface, stomata,glandular trichomes, and leaf hairs.

FIG. 1B: VHH3E6 5 μg/ml in PBS binding to native potato leaf surface.Detection with anti-histidine antibodies directly conjugated withAlexa-488 fluorescent dye; Imaging with a Leica SP5 confocal microscopesystem. VHH 3E6 is binding leaf surface, stomata, glandular trichomes,and leaf hairs.

FIG. 1C: VHH5D4 5 μg/ml in PBS binding to native potato leaf surface.Detection with anti-histidine antibodies directly conjugated withAlexa-488 fluorescent dye. VHH 5D4 is binding leaf surface, stomata,glandular trichomes, and leaf hairs.

FIG. 1D: CBM3a 5 μg/ml in PBS binding to wounded plant tissue on theedge of a potato leaf disc. Detection with anti-histidine antibodiesdirectly conjugated with Alexa-488 fluorescent dye. CBM3a is not bindingleaf surface, stomata, glandular trichomes, or leaf hairs, but onlybinding to wounded plant tissue on the edge of a potato leaf disc thatis exposed from preparing the sample by punching the leaf.

FIG. 1E: Without primary antibody (plain PBS) on native potato leafsurface. Incubation with anti-histidine antibodies directly conjugatedwith Alexa-488 fluorescent dye.

FIG. 1F: VHH3E6 5 μg/ml in PBS binding to native black nightshade leafsurface. Detection with anti-histidine antibodies directly conjugatedwith Alexa-488 fluorescent dye. VHH 3E6 is binding leaf surface,glandular trichomes, and leaf hairs.

FIG. 1G: VHH3E6 5 μg/ml in PBS binding to native grass leaf surface.Detection with anti-histidine antibodies directly conjugated withAlexa-488 fluorescent dye. VHH 3E6 is binding to leaf surface andwounded plant tissue on the edge of a potato leaf disc that is exposedfrom preparing the sample by punching the leaf.

FIGS. 2A and 2B: Binding of binding domains (VHH) to intact livingplant.

FIG. 2A: VHH3E6 5 μg/ml in PBS binding to an intact living plant. Leavesattached to a potato pot plant were submerged in a solution of VHH 3E6.Leaves were sampled. Detection with anti-histidine antibodies directlyconjugated with Alexa-488 fluorescent dye. VHH 3E6 is binding leafsurface, stomata, glandular trichomes, and leaf hairs.

FIG. 2B: VHH3E6 5 μg/ml in PBS binding to an intact living plant. Leavesattached to a potato pot plant were submerged in a solution of VHH 3E6.Leaves were sampled. Detection with anti-histidine antibodies directlyconjugated with Alexa-488 fluorescent dye. Excerpt from whole leaflabeling. VHH 3E6 is binding leaf surface, stomata, glandular trichomes,and leaf hairs.

FIGS. 3A-3D: Coupling of binding domains to microcapsules.

FIG. 3A: Microcapsules with coupled VHH3E6 through one-step EDC couplingchemistry. Coupled microcapsules were labeled with anti-histidineantibodies directly conjugated with Alexa-488 fluorescent dye. Imagingwith a Leica SP5 confocal microscope system. VHH 3E6 is coupled to themicrocapsule surface through one-step coupling chemistry.

FIG. 3B: Microcapsules with coupled VHH3E6 through two-step EDC/NHScoupling chemistry. Coupled microcapsules were labeled withanti-histidine antibodies directly conjugated with Alexa-488 fluorescentdye. Imaging with a Leica SP5 confocal microscope system. VHH 3E6 iscoupled to the microcapsule surface through two-step EDC/NHS couplingchemistry.

FIG. 3C: Microcapsules incubated with VHH3E6 without covalent coupling.Passively adsorbed VHH were labeled with anti-histidine antibodiesdirectly conjugated with Alexa-488 fluorescent dye. Imaging with a LeicaSP5 confocal microscope system. A minor fraction of VHH 3E6 is passivelyadsorbed to the microcapsule surface.

FIG. 3D: Control condition with microcapsules not incubated with VHH butonly with anti-histidine antibodies directly conjugated with Alexa-488fluorescent dye. Imaging with a Leica SP5 confocal microscope system. Aminor fraction of VHH 3E6 is passively adsorbed to the microcapsulesurface.

FIG. 4: Binding and retention of microcapsules to leaf surface. Leafdisc binding assay on native potato leaf discs with microcapsulescontaining a fluorescent tracer molecule. Binding and retention ofmicrocapsules coupled with specific plant-binding VHH, coupled withunrelated control VHH, or blank microcapsules is compared. Nine-foldmore microcapsules coupled with specific VHH are binding and retained onpotato leaf discs compared to blank microcapsules.

FIG. 5: Reduction of dosis using microcapsules coupled with targetingagents. Leaf disc binding assay on native potato leaf discs withmicrocapsules containing a fluorescent tracer molecule. Binding andretention of microcapsules in different concentrations and coupled withspecific plant-binding VHH, coupled with unrelated control VHH, or blankmicrocapsules is compared. Up to eight-fold more microcapsules coupledwith specific VHH are binding and retained on potato leaf discs comparedto blank microcapsules.

DETAILED DESCRIPTION OF THE DISCLOSURE Examples Example 1: Generationand Selection of VHH

Immunization of Llamas with Gum Arabic, Potato Leaf Homogenate, or WheatLeaf Homogenate

A solution of gum arabic was prepared by weighing 5 g of gum arabic fromacacia tree (Sigma) and dissolving in 50 ml water. Bradford proteinassay was used to determine the total protein concentration. Aliquotswere made, stored at −80° C., and used for immunization.

Homogenized leaves from potato plants (Solanum tuberosum varietyDésirée) or wheat plants (Triticum aestivum variety Boldus) wereprepared by freezing leaves in liquid nitrogen and homogenizing theleaves with mortar and pestle until a fine powder was obtained. Bradfordprotein assay was used to determine the total protein concentration.Aliquots were made, stored at −80° C., and suspensions were used forimmunization.

Llamas were immunized at weekly intervals with six intramuscularinjections of gum arabic, homogenized potato leaves, or homogenizedwheat leaves, according to standard procedures. Two Llamas, “404334” and“Lahaïana,” were immunized with gum arabic. Three llamas, “407928,”“Chilean Autumn,” and “Niagara,” were immunized with homogenized potatoleaves and another two llamas, “33733” and “Organza,” were immunizedwith homogenized wheat leaves. Llamas “404334,” “407928,” and “33733”were immunized using Adjuvant LQ (Gerbu), and llamas “Lahaïana,”“Chilean Autumn,” “Niagara” and “Organza” were immunized using Freund'sIncomplete Adjuvant (FIA). Doses for immunization of llama “404334” were350 μg for each day 0, 7, 14, 21, 28, 35, and peripheral bloodlymphocytes (PBL) were collected at day 40. Doses for immunizations ofllamas “407928” and “33733” were 1 mg for each day 0, 7, 14, 21, 28, 36,and PBL were collected at day 40. At time of PBL collection at day 40,sera of llamas “404334,” “407928,” and “33733” were collected. Doses forimmunizations of llamas “Lahaïana,” “Chilean Autumn,” “Niagara,” and“Organza” were 100 μg for day 0, and 50 μg for days 7, 14, 21, 28, and35. At day 0, day 25, and at time of PBL collection at day 38, sera ofllamas “Lahaïana,” “Chilean Autumn,” “Niagara,” and “Organza” werecollected.

Library Construction

From each immunized llama a separate VHH library was made. RNA wasisolated from peripheral blood lymphocytes, followed by cDNA synthesisusing random hexamer primers and Superscript III according to themanufacturer's instructions (Invitrogen). A first PCR was performed toamplify VHH and VH using a forward primer mix [1:1 ratio of call001(5′-gtcctggctgctcttctacaagg-3′ (SEQ ID NO:43)) and call001b(5′-cctggctgctcttctacaaggtg-3′ (SEQ ID NO:44))] and reverse primercall002 (5′-ggtacgtgctgttgaactgttcc-3′ (SEQ ID NO:45)). After isolationof the VHH fragments a second PCR was performed using forward primer A6E(5′-gatgtgcagctgcaggagtctggrggagg-3′ (SEQ ID NO:46)) and reverse primer38 (5′-ggactagtgcggccgctggagacggtgacctgggt-3′ (SEQ ID NO:47)). The PCRfragments were digested using PstI and Eco91I restriction enzymes(Fermentas), and ligated upstream of the pIII gene in vector pMES4(GenBank: GQ907248.1). The ligation products were ethanol precipitatedaccording to standard protocols, resuspended in water, andelectroporated into TG1 cells. Library sizes ranged from 1E+08 to 6E+08independent clones. Single colony PCR on randomly picked clones from thelibraries was performed to assess insert percentages of the libraries.All libraries had ≧90% insert percentages except for the library fromimmunized llama “Organza” which had an insert percentage of 80%.Libraries were numbered 25, 27, 28, 29, 30, 31, 32 for llamas “404334,”“407928,” “33733,” “Chilean Autumn,” “Lahaïana,” “Niagara,” and“Organza,” respectively. Phage from each of the libraries were producedusing VCSM13 helper phage according to standard procedures.

Phage Selections Against Gum Arabic, Plant Epidermal Extracts, or WholeLeaves.

A solution of gum arabic was prepared by weighing 5 g of gum arabic anddissolving in 50 ml water. Aliquots were made and stored at −20° C.until use.

Extracts of potato plant cuticle and adhering epidermis were preparedfrom thin strips from stems of potato plants. Extracts of wheat plantcuticle and adhering epidermis were prepared from thin strips from wheatsheath leaves. Extracts enriched in cell-wall glycans and non-cellulosicpolysaccharides were sequentially extracted using CDTA and NaOH (Molleret al., 2007), respectively. Strips were frozen in liquid nitrogen andground with mortar and pestle until fine powders were obtained.Cell-wall glycans-enriched extracts were prepared by resuspending thefine powders in 50 mM CDTA pH 6.5 using 10 ml per gram of groundmaterial and head-over-head rotation at 4° C. for 30 minutes. Extractand insoluble material were separated using a syringe adapted with afilter. The extracts were further cleared by centrifugation in a microcentrifuge at 20,000 g for 5 minutes. Non-cellulosicpolysaccharide-enriched extracts were prepared from the insolublematerial after CDTA extraction in 4 M NaOH and 1% NaBH₄ using 10 ml pergram of insoluble material and head-over-head rotation at 4° C. for 30minutes. Extract and insoluble material were separated using a syringeadapted with a filter. The extracts were further cleared bycentrifugation in a micro centrifuge at 20,000 g for 5 minutes.

First round selections against gum arabic were performed in wells of a96-well plate (Maxisorp, Nunc) coated with 1 mg/ml or 10 μg/ml gumarabic in 0.1 M carbonate buffer pH 8.3. Coatings were performed at 4°C. overnight. Wells were washed three times with PBS/0.05%-TWEEN®-20 andblocked with 5% skimmed milk in PBS (5% MPBS). Phage were suspended in2.5% MPBS and approximately 2E+11 cfu were used for each well. Afterbinding to the wells at room temperature for 2 hours, unbound phage wereremoved by extensive washing with PBS/0.05%-TWEEN®-20 and PBS. Boundphage were eluted at room temperature with 0.1 mg/ml trypsin (Sigma) inPBS for 30 minutes. Eluted phage were transferred to a polypropylene96-well plate (Nunc) containing excess AEBSF trypsin inhibitor (Sigma).The titers of phage from target-coated wells were compared to titers ofphage from blank wells to assess enrichments. Phage were amplified usingfresh TG1 cells according to standard procedures.

The second selection round was performed similarly to the firstselection round except that for libraries 25 and 30 wells were coatedwith 10 μg/ml and 0.1 μg/ml gum arabic instead of 1 mg/ml and 10 μg/ml.No significant enrichments were obtained for libraries 27, 28, 29, 31,and 32 in selection round 1. In selection round 2, enrichmentswere >1000-fold for libraries 28, 31, and 32, and 25-fold and 250-foldfor libraries 27 and 29, respectively. Enrichments for libraries 25 and30 were 50-fold and >1000-fold in selection round 1, respectively. Inselection round 2, enrichments were 1000-fold for both libraries.Selections against potato epidermal CDTA extract were performedsimilarly to the selections against gum arabic but wells were coatedwith ten-fold and 1000-fold diluted potato epidermal CDTA extract forboth the first and second selection rounds. Enrichments in selectionround 1 were 10, 1E+03, 20, 20, >1E+04, 15, and five-fold for libraries25, 27, 28, 29, 30, 31, 32, respectively and >100-fold for all librariesin selection round 2. Selections against wheat epidermal CDTA extractwere performed similarly to the selections against potato epidermal CDTAextract but wells were coated with 20-fold and 2000-fold diluted wheatepidermal CDTA extract for both the first and second selection rounds.Enrichments in selection round 1 were >10, >100, >10, 1, >1E+03, 10, andfive-fold for libraries 25, 27, 28, 29, 30, 31, 32, respectively.Enrichments in selection round 2 were >ten-fold for library 29and >100-fold for libraries 25, 27, 28, 30, 31, and 32. Selectionsagainst potato leaves were performed in two consecutive selection roundsusing leaf particles in round 1 and whole leaves in round 2. Libraries27, 28, 29, 30, 31, and 32 were used for selections against leaves. Theleaf particles for first round selections were prepared by blendingpotato leaves in PBS using an Ultra-Turrax T25 homogenizer. The leafparticles were collected from the suspension by centrifugation. Thesupernatant, called herein “homogenized leaf soluble fraction,” isassumingly enriched in intracellular components and was used in solutionduring phage selection to compete out binders to intracellular epitopes.Library phage were pre-incubated with the homogenized leaf solublefraction in 2% MPBS using head-over-head rotation at room temperaturefor 30 minutes. The mixtures were added to leaf particles and incubatedwith head-over-head rotation at room temperature for 2 hours. Leafparticles with bound phage were collected by centrifugation andsupernatants were discarded. Leaf particles with bound phage were washedextensively by consecutive washes with PBS. Washes were performed byresuspending leaf particles in PBS, spinning down leaf particles, anddiscarding supernatants. Elution of phage and infection of TG1 wereperformed as before. For the second selection round whole intact leaveswere used. Leaves were incubated floating upside-down on phage solutionsin 2% MPBS and phage were allowed to bind at room temperature for 2hours. The leaves were washed extensively by transferring leaves tofresh tubes with PBS. Elution of bound phage was performed with 100 mMTEA in water, and solutions with eluted phage were neutralized usinghalf of the eluted phage volume of 1 M Tris pH 7.5. Infection of TG1 wasperformed as before.

Picking Single Colonies From Selection Outputs

Individual clones were picked from first and second round selectionsagainst gum arabic with libraries 25 and 30. From selections against gumarabic with libraries 27, 28, 29, 31, and 32, clones were picked aftersecond round selections but not first round selections. A total of 208clones was picked from gum arabic selections. From selections againstpotato epidermal CDTA extract a total of 321 clones was picked afterboth first and second round selections from all libraries. Fromselections against wheat epidermal CDTA extract a total of 162 cloneswas picked after second round selections from all libraries. From potatoleaf selections a total of 184 clones was picked after second roundselections from libraries 27, 28, 29, 30, 31, and 32. Fresh TG1 cellswere infected with serially diluted eluted phage and plated on LB agar;2% glucose; 100 μg/ml ampicillin. Single colonies were picked in 96-wellplates containing 100 μl per well 2×TY; 10% glycerol; 2% glucose; 100μg/ml ampicillin. Plates were incubated at 37° C. and stored at −80° C.as master plates.

Example 2: Characterization of the VHH Single-Point Binding ELISA

A single-point binding ELISA was used to identify clones that bind togum arabic or plant extracts. VHH-containing extracts for ELISA wereprepared as follows. 96-well plates with 100 μl per well 2×TY, 2%glucose 100 μg/ml ampicillin were inoculated from the master plates andgrown at 37° C. overnight. 25 μl per well of overnight culture was usedto inoculate fresh 96-well deep-well plates containing 1 ml per well2×TY; 0.1% glucose; 100 μg/ml ampicillin. After growing at 37° C. in ashaking incubator for 3 hours, IPTG was added to 1 mM finalconcentration and recombinant VHH was produced during an additionalincubation for 4 hours. Cells were spun down by centrifugation at 3,000g for 20 minutes and stored at −20° C. overnight. Cell pellets werethawed, briefly vortexed, and 125 μl per well of room temperature PBSwas added. Cells were resuspended on an ELISA shaker platform at roomtemperature for 15 minutes. Plates were centrifuged at 3,000 g for 20minutes and 100 μl per well of VHH-containing extract was transferred topolypropylene 96-well plates (Nunc) and stored at −20° C. until furtheruse.

Binding of clones from gum arabic selections was analyzed in ELISAplates coated with 100 μl/well gum arabic at 1 mg/ml in carbonate bufferpH 8.3. Binding of clones from potato epidermal CDTA extract selectionswas analyzed on both potato epidermal CDTA extract and wheat epidermalCDTA extract using ELISA plates coated with 100 μl per well of 30-folddiluted potato and 30-fold wheat epidermal CDTA extracts in 0.1 Mcarbonate pH 8.3. Binding of clones from wheat epidermal CDTA extractselections was analyzed using ELISA plates coated with 100 μl per wellof 20-fold diluted wheat epidermal CDTA extract in 0.1 M carbonate pH8.3. After coating at 4° C. overnight and continued coating at roomtemperature for 1 hour on the next day, plates were washed three timeswith PBS/0.05%-TWEEN®-20 and blocked with 5% skimmed milk in PBS for 1.5hours. Plates were emptied and filled with 90 μl per well 1% MPBS. 10 μlof VHH-containing extract from each clone was added to (an)antigen-coated well(s) and a blank well. VHH were allowed to bind atroom temperature for 1 hour and unbound VHH were removed by washingthree times with PBS/0.05%-TWEEN®-20. Bound VHH were detected withsequential incubations with monoclonal mouse anti-histidine antibodies(Abd Serotec) in 1% MPBS/0.05%-TWEEN®-20 and rabbit anti-mouse IgG wholemolecule antibodies conjugated with alkaline phosphatase (RaM/AP)(Sigma) in 1% MPBS/0.05%-TWEEN®-20. Unbound antibodies were removed bywashing three times with PBS/0.05%-TWEEN®-20. The plates were washed anadditional two times with PBS and 100 μl pNPP disodium hexahydratesubstrate (Sigma) was added to each well.

The absorbance at 405 nm was measured and the ratio of VHH bound to (a)target-coated well(s) and a non-target-coated well was calculated foreach clone. 23% of clones had a ratio greater than 2 and these cloneswere firstly picked for more detailed characterization. A second groupof clones with a ratio between 1.15 and 2, and comprising 10% of allclones, was revisited later. Clones with a ratio less than 1.15 were notanalyzed further.

For clones from whole leaf selections an adapted ELISA was developed.Upside-down floating leaf discs were used instead of coating wells withantigen. Incubations were similar to the extracts ELISA. Afterincubation with the substrate the leaf discs were removed from the wellsusing a forceps and the absorbance at 405 nm was measured. Signalsobtained for each clone were compared to signals obtained from wellswith leaf discs without primary antibody incubation and the ratios werecalculated. A leaf surface-binding antibody that was found andcharacterized from epidermal extract selections was used as positivecontrol antibody. VHH with a ratio greater than 1.5 were analyzedfurther by sequencing.

Single Colony PCR and Sequencing

Single colony PCR and sequencing was performed on ELISA positive clonesas follows. Cultures from master plate wells with ELISA positive cloneswere diluted ten-fold in sterile water. 5 μl from these diluted cloneswere used as template for PCR using forward primer MP57(5′-ttatgcttccggctcgtatg-3′ (SEQ ID NO:48)) and reverse primer Gill(5′-ccacagacagccctcatag-3′ (SEQ ID NO:49)). PCR products were sequencedby Sanger-sequencing using primer MP57 (VIB Genetic Service Facility,University of Antwerp, Belgium).

Antibody Production and Purification

VHH antibody fragments were produced in E. coli suppressor strain TG1 ornon-suppressor strain WK6 (Fritz et al., Nucleic Acids Research, Volume16 Number 14 1988) according to standard procedures. Briefly, colonystreaks were made and overnight cultures from single colonies inoculatedin 2×TY; 2% glucose; 100 μg/ml ampicillin. The overnight cultures wereused to inoculate fresh cultures 1:100 in 2×TY; 0.1% glucose; 100 μg/mlampicillin. After growing at 37° C. in a shaking incubator for 3 hours,IPTG was added to a 1 mM final concentration and recombinant VHHantibody fragments were produced during an additional incubation for 4hours. Cells were spun down and resuspended in 1/50^(th) of the originalculture volume of periplasmic extraction buffer (50 mM phosphate pH 7; 1M NaCl; 1 mM EDTA) and incubated with head-over-head rotation at 4° C.overnight. Spheroplasts were spun down by centrifugation at 3,000 g and4° C. for 20 minutes. Supernatants were transferred to fresh tubes andcentrifuged again at 3,000 g and 4° C. for 20 minutes.Hexahistidine-tagged VHH antibody fragments were purified from theperiplasmic extract using 1/15^(th) of the extract volume of TALON metalaffinity resin (Clontech), according to the manufacturer's instructions.Purified VHH antibody fragments were concentrated and dialyzed to PBSusing Vivaspin 5 kDa MWCO devices (Sartorius Stedim), according to themanufacturer's instructions.

VHH Binding to Gum Arabic in ELISA

Titration of VHH antibody fragments was performed on ELISA plates(Maxisorp, Nunc) coated with 100 μl per well 100 μg/ml gum arabic in 50mM carbonate pH 9.6. Plates were coated at 4° C. overnight and coatingwas continued at room temperature for 1 hour on the next day. Plateswere washed three times with PBS/0.05%-TWEEN®-20 and blocked with 5%skimmed milk in PBS for 1 hour. Four-fold serial dilutions of purifiedVHH antibody fragments were prepared in 1% MPBS/0.05%-TWEEN®-20 inpolypropylene 96-well plates. Antibody concentrations ranged from 3μg/ml to 12 ng/ml. Antibody dilutions were transferred to the gumarabic-coated plates and VHH antibody fragments were allowed to bind for1 hour at room temperature. Bound VHH were detected with sequentialincubations with monoclonal mouse anti-histidine antibodies (AbdSerotec) and rabbit anti-mouse IgG whole molecule antibodies conjugatedwith alkaline phosphatase (RaM/AP) (Sigma) in 1% MPBS/0.05%-TWEEN®-20.Unbound antibodies were removed by washing three times withPBS/0.05%-TWEEN®-20 after each antibody incubation. The plates werewashed an additional two times with PBS and 100 μl pNPP disodiumhexahydrate substrate (Sigma) was added to each well. The absorbance at405 nm was measured and plotted as function of antibody concentration(see Table 1).

VHH Binding to Potato Lectin in ELISA

ELISA plates (Maxisorp, Nunc) coated with 100 μl per well 100 μg/mlpotato lectin (Sigma) in PBS were coated at 4° C. overnight and coatingwas continued at room temperature for 1 hour on the next day. Plateswere washed three times with PBS/0.05%-TWEEN®-20 and blocked with 5%skimmed milk in PBS for 1 hour. VHH (3 μg/ml) were transferred to thepotato lectin-coated plates and VHH antibody fragments were allowed tobind for 1 hour at room temperature. Bound VHH were detected withsequential incubations with monoclonal mouse anti-histidine antibodies(Abd Serotec) and rabbit anti-mouse IgG whole molecule antibodiesconjugated with alkaline phosphatase (RaM/AP) (Sigma) in 1%MPBS/0.05%-TWEEN®-20. Unbound antibodies were removed by washing threetimes with PBS/0.05%-TWEEN®-20 after each antibody incubation. Theplates were washed an additional two times with PBS and 100 μl pNPPdisodium hexahydrate substrate (Sigma) was added to each well and theabsorbance at 405 nm was measured (see Table 2).

TABLE 2 VHH VHH VHH VHH VHH 3E6 5D4 5C4 5G5 7D2 <Blank Gum arabic 0.8820.530 0.873 0.751 0.274 0.069 Potato lectin 4.000 4.000 4.000 4.0004.000 0.081 Blank 0.067 0.072 0.071 0.073 0.072 0.072

Example 3: Binding of Binding Domains to Plant Surface VHH Binding toLeaf Discs

VHH binding to non-fixed leaf discs of potato (variety Désirée), blacknightshade, grass, wheat or azalea was investigated. For comparison,binding of CBM3a to non-fixed leaf discs of potato (variety Désirée) wasanalyzed in parallel. Leaf discs were prepared by punching a freshpotato leaf with a 5 mm belt hole puncher tool. Leaf discs were putimmediately in wells of a 96-well plate containing 200 μl per well 5%MPBS or PBS, and incubated for 30 minutes. Leaf discs were transferredto solutions containing 5 μg/ml VHH antibody fragment, respectively, 5μg/ml CBM3a in 2% MPBS or PBS and incubated for 60-90 minutes. UnboundVHH or CBM3a proteins were removed by washing three times with 2% MPBSor PBS. Bound VHH or CBM3a proteins were detected with incubation withmonoclonal mouse anti-histidine antibodies directly conjugated withAlexa-488 fluorescent dye (Abd Serotec) in 1% MPBS for 1 hour. Unboundantibodies were removed by washing three times with PBS. Leaf discs wereput on glass slides, covered with cover slips, and analyzed bymicroscopy or on a macrozoom microscope system (Nikon) or a SP5 confocalmicroscope system (Leica). By means of a non-limiting example, VHHantibody fragments (e.g., 3E6, 5D4) were found to be clearly binding totrichomes, stomata and cuticle at the leaf surface of potato leaves(FIGS. 1A-1C). In sharp contrast, for CBM3a no binding at the surface ofpotato leaves was detected and only faint binding to the wound tissue atthe cut edge of the potato leaf disc was observed (FIG. 1D). Some VHHhereof (e.g., 3E6) were also shown to bind specifically to the surfaceof black nightshade leaves or grass leaves or as shown in FIGS. 1F and1G, respectively. No significant binding was observed to the leafsurface of wheat or azalea.

VHH Binding to Intact Living Plants

Binding of VHH to intact living plants was investigated on potato potplants (variety Desirée). Compound leaves of intact living plants weresubmersed in solutions of hexahistidine-tagged VHH in PBS, or PBS alonefor control conditions, leaving the compound leaves attached to theplants. VHH were allowed to bind for 1 hour. Next, the compound leavesstill attached to the plants were washed five times in PBS in Erlenmeyerflasks. Different leaves and petiole sections were sampled. Bound VHHwere detected by incubation with monoclonal mouse anti-histidineantibodies directly conjugated with Alexa-488 fluorescent dye (AbdSerotec) in PBS for 1 hour. Unbound anti-histidine antibodies wereremoved by washing five times with PBS. Whole leaves, leaf discs, orpetiole sections were analyzed for bound VHH with microscopy. VHH provedto bind leaf structures such as trichomes and stomata, leaf surface, andpetiole sections as shown in FIGS. 2A and 2B. No binding was observedwith unrelated control VHH, proving that the VHH hereof are capable ofspecifically binding to intact living plants.

VHH Binding in Water

Binding of VHH to leaf surfaces in water was investigated on leaf discscut from leaves from potato plants (variety Desirée). Leaf discs werewashed three times in ultrapure water. Hexahistidine-tagged VHH werediluted in ultrapure water, added to leaf discs, and allowed to bind for1 hour. Although the stock solutions of VHH were in PBS, the dilutionsused here (200-fold for 5 μg/ml, or 2000-fold for 500 ng/ml) result insignificant dilution of PBS from the stocks and can be consideredsufficiently dilute to represent binding in water. After allowing VHH tobind for 1 hour, leaf discs were washed five times with ultrapure water.Bound VHH were detected by incubation with monoclonal mouseanti-histidine antibodies directly conjugated with Alexa-488 fluorescentdye (Abd Serotec) in PBS for 1 hour. Unbound anti-histidine antibodieswere removed by washing five times with PBS. Leaf discs were analyzedfor bound VHH with microscopy. Binding of VHH in PBS was analyzed asdescribed before as a control condition. Detection of bound VHH withanti-histidine antibodies conjugated with Alexa-488 fluorescent dye,washing away non bound anti-histidine antibodies, and analyzing boundVHH with microscopy was performed as for the VHH binding experiment inwater. VHH proved to bind in water to leaf structures such as trichomesand stomata, and leaf surface. No binding was observed with unrelatedcontrol VHH. The observed binding in water was similar as seen for theparallel experiment performed in PBS. The VHH hereof are capable ofbinding leaf structures and leaf surface in water.

VHH Binding Kinetics

In order to further test applicability of VHH as binders for greenhouseor field applications where binding supposedly needs to be achievedquickly after application, a leaf dip VHH binding experiment wasemployed to test minimum incubation times of VHH to achieve detectablebinding. ø8 mm potato leaf discs (variety Desirée) were cut using apuncher tool and washed three times in PBS. 5 μg/ml pre-dilutions ofhexahistidine-tagged VHH were prepared in PBS and incubated fordifferent times with the leaf discs. The times for incubation were 10seconds, 30 seconds, 1 minute, 5 minutes, 20 minutes, or 1 hour. UnboundVHH were removed by washing five times with PBS. Bound VHH were detectedby incubation with monoclonal mouse anti-histidine antibodies directlyconjugated with Alexa-488 fluorescent dye (Abd Serotec) in PBS for 1hour. Unbound anti-histidine antibodies were removed by washing fivetimes with PBS. Leaf discs were analyzed for bound VHH with microscopy.Specific binding was observed for each sample with specific VHH fromincubation time 10 seconds to VHH incubation time 1 hour. No binding wasobserved with unrelated control VHH. The VHH hereof show detectablebinding to leaf structures, such as trichomes and stomata and leafsurface within 10 seconds after application.

VHH Binding at Different pH

In order to test applicability of VHH as binders for greenhouse or fieldapplications where binding supposedly may occur at pH-values, deviatingstrongly from physiological conditions in which antibodies naturallybind their targets, a leaf dip VHH binding experiment was carried out ina series of solutions with different pH. The following solutions wereprepared: 50 mM glycine pH 2.0, 50 mM sodium acetate pH 4.0, 50 mmsodium carbonate pH 9.6, and 10 mM sodium hydroxide pH 11.0. 0 8 mmpotato leaf discs (variety Desirée) were cut using a puncher tool. Theleaf discs were first equilibrated to the different pH by washing threetimes with solutions at different pH. Hexahistidine-tagged VHH werediluted to 5 μg/ml in solutions with different pH, added to thecorresponding equilibrated leaf discs, and binding of VHH was allowedfor 1 hour. After incubation with VHH, leaf discs were washed threetimes with solutions at the corresponding different pH. After that, allwere washed two times with PBS to equilibrate leaf discs to PBS. BoundVHH were detected by incubation with monoclonal mouse anti-histidineantibodies directly conjugated with Alexa-488 fluorescent dye (AbdSerotec) in PBS for 1 hour. Unbound anti-histidine antibodies wereremoved by washing five times with PBS. Leaf discs were analyzed forbound VHH with microscopy. Some of the VHH hereof (e.g., VHH 3E6) showeddetectable binding to leaf discs over the whole range tested from pH 2to pH 11.

VHH Binding at Different Temperatures

In order to test applicability of VHH as binders for greenhouse or fieldapplications where binding supposedly may occur at different andsometimes even extreme temperatures, a leaf dip VHH binding experimentat different temperatures was used. Temperatures used were 4° C., roomtemperature, 37° C., 55° C., or 70° C. ø8 mm potato leaf discs (varietyDesirée) were cut using a puncher tool. The leaf discs were equilibratedto different temperatures by washing three times with PBS at differenttemperatures. Hexahistidine-tagged VHH were diluted to 5 μg/ml in PBS atdifferent temperatures, added to the corresponding equilibrated leafdiscs, and binding of VHH was allowed for 1 hour at differenttemperatures. After incubation with VHH, leaf discs were washed fivetimes with PBS at room temperature. Bound VHH were detected byincubation with monoclonal mouse anti-histidine antibodies directlyconjugated with Alexa-488 fluorescent dye (Abd Serotec) in PBS for 1hour at room temperature. Unbound anti-histidine antibodies were removedby washing five times with PBS at room temperature. Leaf discs wereanalyzed for bound VHH with microscopy. Some of the VHH hereof (e.g.,VHH 3E6) showed detectable binding to leaf discs over a temperaturerange from 4° C. to 55° C. Please note that leaf discs severely sufferwhen submerged in PBS at 70° C. for 1 hour but that binding of VHH wasstill detected.

Example 4: Coupling of Targeting Agents to Microparticles Construction,Production and Purification of Bivalent VHH

Bivalent VHH constructs were produced in bacteria by cloning two VHHsequences in tandem into the pASF22 vector, creating a fusion of two VHHwith a 9 glycine-serine linker (GGGGSGGGS (SEQ ID NO:50)) in between thetwo VHH. pASF22 is an in-house produced pMES derivative. The tags thatwere used were C-terminal c-Myc (EQKLISEEDLN (SEQ ID NO:51)) andhexahistidine (HHHHHH (SEQ ID NO:52)). A triple alanine linker (AAA) wasplaced in between the C-terminal end of the VHH and the c-Myc tag and aglycine-alanine-alanine (GAA) linker was used in between the C-terminalend of the c-Myc tag and the hexahistidine tag. The complete sequenceC-terminal of the bivalent VHH that was used: AAA-EQKLISEEDLN-GAA-HHHHHH(SEQ ID NO:53). Fresh overnight cultures were produced by starting fromcolony streaks and inoculation of 2xTY media supplemented with 2%glucose and 100 μg/ml ampicillin. The overnight cultures were used toinoculate fresh cultures 1:100 in 2×TY media with 0.1% glucose and 100μg/ml ampicillin. After growing at 37° C. in a shaking incubator for 3hours, IPTG was added to a 1 mM final concentration and recombinantbivalent VHH were produced during an additional incubation for 4 hours.Cells were spun down and resuspended in 1/50th of the original culturevolume of PBS and incubated with head-over-head rotation at 4° C. for 30minutes. Spheroplasts were spun down by centrifugation at 3,000 g and 4°C. for 20 minutes. Supernatants were transferred to fresh tubes andcentrifuged again at 3,000 g and 4° C. for 20 minutes. The supernatantwas collected and sodium chloride concentration was adjusted to 500 mMand imidazole concentration to 20 mM. Hexahistidine-tagged bivalent VHHwere purified from the extracts using HisTrap FF Crude 5 ml IMAC columns(GE Lifesciences) and HiLoad 16/60 Superdex 75 prep grade gel filtrationcolumn (GE Lifesciences) on an AKTAxpress system (GE Lifesciences)following standard procedures.

Coupling of VHH to Microparticles

It was first examined whether VHH that are covalently bound tomicroparticles can bind their target and provide sufficient adhesionstrength to a surface containing antigen for targeting of themicroparticle. Microparticles were coupled with gum arabic-specific VHHantibody fragments and binding to ELISA plates coated with gum arabicwas investigated.

Different types of microparticles were prepared. Purified VHH antibodyfragments were (i) coupled to Ø2.8 μm paramagnetic Dynabeads M-270carboxylic acid (Dynal, Invitrogen), using a two-step coupling chemistryof EDC activation of the beads and subsequent coupling of VHH antibodyfragments, and (ii) coupled using a one-step coupling chemistry to Ø2 μmFluoSpheres fluorescent microspheres (Molecular Probes, Invitrogen),both according to the manufacturers' instructions.

Briefly, for coupling to Dynabeads M-270 carboxylic acid: VHH weredialyzed to 50 mM MES buffer pH 5.0 using Vivaspin 5 kDa spin filterdevices (Sartorius Stedim). Beads were prepared by two sequential washeswith 10 mM NaOH, and three washes with water, and activated with 0.1 MEDC (Pierce) at room temperature for 30 minutes. EDC-activated beadswere washed by quick sequential washes with ice-cold water and ice-cold50 mM MES buffer pH 5.0. Beads were dispensed with the last wash. 60 μgof VHH antibody fragment in 100 μl 50 mM MES pH 5.0 were added to 3 mgbeads and incubated at room temperature for 30 minutes. The supernatantafter coupling was collected. By measuring protein A280 of the non-boundfraction the amounts of coupled and non-coupled VHH were calculated.Greater than 95% of VHH antibody fragment were coupled to the beads.Beads were blocked with 50 mM Tris pH 7.4 and washed three times withPBS/0.1%-TWEEN®-20 and stored at 4° C.

Briefly, for coupling to FluoSpheres fluorescent microspheres: VHH weredialyzed to 50 mM MES buffer pH 6.0 using Vivaspin 5 kDa spin filterdevices (Sartorius Stedim). 0.8 μm PES filter devices (Sartorius Stedim)were used throughout the procedure to isolate beads from solution. Beadswere prepared by washing with ultrapure water and re-suspension inultrapure water. 100 μl of VHH antibody fragments containing 200 μg VHHwere added to 100 μl beads. 0.8 mg EDC (Pierce) was added to each mix ofbeads with VHH and the pH was adjusted to 6.5 with 0.1 M NaOH. Couplingwas performed at room temperature for 2 hours. Glycine was added to afinal concentration of 100 mM and incubated at room temperature for 30minutes to quench the reaction. By measuring protein A280 of thenon-bound fraction the amounts of coupled and non-coupled VHH werecalculated. Between 14% and 33% of different VHH antibody fragments werecoupled to the beads. Beads were washed twice with 50 mM phosphate pH7.4; 0.9% NaCl (50 mM PBS) and stored in 1% BSA, 2 mM sodium azide in 50mM PBS.

Coupling of Targeting Agents to Microcapsules Containing FluorescentTracer or Active Ingredient

Polyurea microcapsules were produced by interfacial polymerization. Withthe objective to generate functionalized polyurea microcapsules, VHHwere coupled to microcapsules containing either the insecticide lambdacyhalothrin or the fluorescent tracer molecule Uvitex OB and a shellwith incorporated lysine to surface-expose carboxylic acid residues.Lambda cyhalothrin was dissolved in benzyl benzoate in concentrationsbetween 30% and 66% before encapsulation. Alternatively, a core of 1.5%Uvitex in benzyl benzoate was used for easy fluorescent visualization ofmicrocapsules. Toluene diisocyanate (TDI) and polymethylenepolyphenyleneisocyanate (PMPPI) were dissolved in the oil phase in different ratiosand concentrations in the oil phase to produce desired shellcharacteristics. Stirring speed for the emulsion was varied to controldroplet size and consequently microcapsule diameter. Microcapsules withapproximate diameters of 5 μm, 10 μm, or 50 μm were successfullyproduced. Bifunctional lysine and trifunctional diethylene triamine(DETA) were used in different ratios and/or added sequentially duringencapsulation to on the one hand maximize amounts of carboxylic acids onthe microcapsules' surface and on the other hand obtain sufficientstrength of capsule shells. Microcapsules were washed with water afterproduction and stored as microcapsule suspensions in water. Themicrocapsules were washed with 100 mM MES, 500 mM NaCl, pH 6.0immediately before coupling of VHH using a vacuum-tight filter flask andP 1.6 filter funnel (Duran). Alternatively, glass filter holders with0.45 μm disposable membrane filters (Millipore) or 0.45 μm 96-welldeep-well filtration plates (Millipore) were used. Couplings of VHH tomicrocapsules were performed using carbodiimide-mediated couplings usinga one-step procedure, a two-step procedure without N-hydroxysuccinimide(NHS), or a two-step procedure with NHS. The major difference betweenone-step coupling and two-step coupling procedures is the occurrence ofcross-linking of VHH in one-step coupling procedures. The protocols forthe three procedures are largely similar and differ as follows. Forone-step couplings VHH were added to washed microcapsules and1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC)(Pierce) was added and coupling reaction was allowed for 2 hours at roomtemperature. For two-step couplings washed microcapsules were firstactivated with EDC in the presence or absence of NHS. Excess unreactedEDC (and NHS) were removed by quick sequential washes with ice-coldbuffers and VHH were added and allowed to react with activatedcarboxylic acids on microcapsule shells. For ø10 μm microcapsules 2-20μg VHH were coupled per mg microcapsules. For microcapsules with otherdiameters amounts were scaled accordingly. After coupling of VHH themicrocapsules were washed with PBS and stored in PBS. Success ofcoupling of VHH was investigated using a combination of analyzingcoupling efficiency by SDS-PAGE and analyzing bound hexahistidine-taggedVHH by microscopy or a SP5 confocal microscope system (Leica) usinganti-histidine antibodies directly conjugated with Alexa-488 fluorescentdye. With SDS-PAGE analysis formation of multimers was observed forone-step coupling reactions as expected. VHH-coupled microcapsules werelabeled with anti-histidine antibodies for 1 hour at room temperature.Unbound anti-histidine antibodies were removed by washing five timeswith PBS using 0.45 μm 96-well deep-well filtration plates (Millipore).Microcapsules with coupled VHH, microcapsules incubated with VHH towhich no EDC was added, and blank microcapsules were compared.Anti-histidine labeling of microcapsules was most intense formicrocapsules to which VHH had been coupled using either one-step ortwo-step coupling procedures as shown in FIGS. 3A-3D. It was alsoobserved that some VHH were passively adsorbed to the microcapsules. VHHwere successfully coupled to microcapsules of different size usingeither one-step or two-step coupling procedures.

Example 5: Binding of Targeting Agent-Coupled Micro Particles toAntigen-Containing Surface

Binding Assays with VHH-Coupled Beads or Microcapsules

Functionality of VHH-coupled microparticles was investigated in ELISAplates that were coated with 100 μg/ml gum arabic in 50 mM carbonate pH9.6 or PBS. Coating was performed overnight and plates were washed threetimes with PBS/0.05%-TWEEN®-20 and blocked with 5% skimmed milk in PBSfor 1.5 hours. VHH-coupled paramagnetic beads were diluted 50-fold andincubated with monoclonal mouse anti-histidine antibodies directlyconjugated with Alexa-488 fluorescent dye (Abd Serotec) in 1% MPBST for1 hour. Two-fold serial dilutions (50- to 800-fold) of VHH-conjugatedparamagnetic Dynabeads and FluoSpheres fluorescent beads were preparedin 2% MPBS, transferred to the gum arabic-coated ELISA plates, andincubated at room temperature for 1 hour. Unbound beads were removed bywashing five times with PBS/0.05%-TWEEN®-20. The bottoms of ELISA platewells were analyzed for bound beads by microscopy. Counting beads andusing the microscope's camera mask for calculation of the analyzedsurface area were used for calculating number of bound beads per well asshown in Table 3. Alternatively, microparticles were visualized using amacrozoom microscope system (Nikon) and counted using Volocity imageanalysis software (PerkinElmer); the number of bound Fluospheres perwell is shown in Table 4.

TABLE 3 Counted bound magnetic carboxylic acid Dynabeads to wells coatedwith gum arabic Magnetic Carboxylic Acid Dynabeads 2.8 μm (approximatenumbers) Dilution Gum arabic Coupled with VHH 3E6 Coupled with VHH 5D450 + ≈1000  ≈500 100 + ≈500 ≈500 200 + ≈200 ≈200 400 + ≈100 ≈200 800 +≈100 ≈100 50 −  ≈10  ≈50

TABLE 4 Counted bound Fluospheres to wells coated with gum arabic Numberof Fluospheres Fluospheres Fluospheres coupled coupled Coating addedwith VHH 3E6 with unrelated VHH No coating 4.5 · 10⁶ 115 198 Gum arabic4.5 · 10⁶ 1874 224 Gum arabic 2.3 · 10⁶ 1273 89 Gum arabic 1.1 · 10⁶ 98183

An ELISA-like assay setup was used to evaluate the interaction ofVHH-coupled microcapsules to antigen-containing surfaces. ELISA plates(Maxisorp (Thermo Scientific Nunc) or high bind half area microplates(Greiner Bio-One)) were coated with gum arabic or potato lectin.Coatings were performed overnight with 100 μg/ml gum arabic or potatolectin in PBS. Control wells included blank wells or wells coated withunrelated antigens. Plates were washed three times with PBS with0.05%-TWEEN®-20 and blocked with 5% skimmed milk in PBS for 1 to 2hours. VHH-coupled lambda cyhalothrin-containing or Uvitex-containingmicrocapsules were diluted to appropriate densities in 1% skimmed milkin PBS with 0.05%-TWEEN®-20. Microcapsules were added to theantigen-coated or control wells and allowed to bind for 1 hour. Unboundmicrocapsules were removed by washing five times with PBS with0.05%-TWEEN®-20. The bottoms of ELISA plate wells were analyzed forbound microcapsules on a macrozoom microscope system (Nikon).Microcapsules were counted using Volocity image analysis software(Perkin Elmer). A DAPI filter was used to visualize Uvitexmicrocapsules. White LED illumination and bright field pictures wereused for lambda cyhalothrin microcapsules. Controls for lambdacyhalothrin-containing or Uvitex-containing microcapsules included blankmicrocapsules and microcapsules to which unrelated VHH were coupled.

TABLE 5 Bound microcapsules to wells coated with potato lectin orunrelated antigen Counts Counts Counts Area Area Microcapsulescontaining Microcapsules lambda-cyhalothrin containing uvitex OB SurfaceBlank unrelated VHH VHH unrelated coverage microcapsules control 3E6 3E6control no coating 100% 583 689 701 86.574 82.757 potato lectin 100% 755828 7.910 504.839 16.676 potato lectin  20% 616 709 4.550 510.242 35.433potato lectin  4% 408 348 798 144.955 7.529 no coating 100% n.d. n.d.209 68.181 60.841 unrelated antigen 100% n.d. n.d. 861 84.508 94.153unrelated antigen  20% n.d. n.d. 601 47.906 39.218 unrelated antigen  4%n.d. n.d. 386 23.525 18.517

In another experiment, lambda cyhalothrin amounts were also determinedanalytically. 100 μl well aceton was added to washed wells with boundmicrocapsules and transferred to glass vials with 10 ml of hexanecontaining 0.05% triphenylphosphate as internal standard. The amount oflambda cyhalothrin was determined by GC/MS-MS analysis in comparisonwith calibration solutions. Controls for lambda cyhalothrinmicrocapsules included blank microcapsules to which no VHH were coupledand microcapsules to which unrelated VHH were coupled. Controls alsoincluded wells to which no gum arabic or potato lectin was coated. Basedon the results of the ELISA-like assay with lambda cyhalothrinmicrocapsules it was found that some of the VHH hereof (e.g., VHH3E6)are capable of binding and retaining microcapsules to antigen-coatedsurfaces resulting in a 23-fold increase of amounts of lambdacyhalothrin in wells coated with antigen compared to blank microcapsulesand a 27-fold increase was measured over blank wells not coated withantigen.

Based on the results of the microcapsule binding assays, VHH could beclassified as capable or not capable of binding and retainingmicrocapsules to a surface. Some of the VHH hereof (e.g., VHH3E6) provedcapable of binding specifically to antigen-coated surfaces when coupledto a microcapsule. No significant binding to surfaces with unrelatedantigens was observed. Moreover, the specific binding was strong enoughto retain the microcapsule at the antigen-coated surface, as the bindingforce clearly resists the shear forces that occur during the washingprocedure. What is more is that VHH are capable of binding and retainingmicrocapsules containing relevant active ingredients to surfaces, asshown, for example, with microcapsules containing the insecticide lambdacyhalothrin.

Next, it was investigated if binding of microcapsules to surfaces couldbe improved by using targeting agents comprising multivalent VHH. Aseries of parallel couplings was performed with equal amounts ofmonovalent VHH, bivalent VHH, and unrelated VHH. Success of coupling ofVHH and multivalent VHH were analyzed as described in Example 4. AnELISA-like assay was performed using high bind half area microplates(Greiner Bio-One) coated with 5 μg/well potato lectin. Control wellsincluded blank wells or wells coated with unrelated antigens. Plateswere washed three times with PBS with 0.05%-TWEEN®-20 and blocked with5% skimmed milk in PBS for 1 to 2 hours. VHH-coupled Uvitex-containingmicrocapsules were diluted to appropriate densities in 1% skimmed milkin PBS with 0.05%-TWEEN®-20. Five-fold serial dilution series wereprepared and allowed to bind to the surface to compare binding ofmicrocapsules coupled with monovalent or bivalent VHH. Microcapsuleswere added to the antigen-coated or control wells and allowed to bindfor 1 hour. Unbound microcapsules were removed by washing five timeswith PBS with 0.05%-TWEEN®-20. The bottoms of ELISA plate wells wereanalyzed for bound microcapsules on a macrozoom microscope system(Nikon). Microcapsules were counted using Volocity image analysissoftware (Perkin Elmer). A DAPI filter was used to visualize Uvitexmicrocapsules. Bivalent VHH proved capable of binding specifically to anantigen-coated surface when coupled to a microcapsule and moremicrocapsules were retained using bivalent VHH compared to microcapsuleswith monovalent VHH. With the highest density of microcapsules applied(calculated to fully cover the surface of the bottom of the well) it wasfound that 17% more microcapsules with coupled bivalent VHH wereretained in the well compared to the same amount of microcapsules withmonovalent VHH. With an application of 25-fold less microcapsules it wasfound that 160% more microcapsules were retained in the well formicrocapsules coupled with bivalent VHH compared to microcapsules withmonovalent VHH. The surface area of microcapsules with coupled bivalentVHH was 15-fold above the surface area of blank microcapsules applied atthis microcapsule density while the surface area of microcapsules withmonovalent VHH was only six-fold above the surface area of blankmicrocapsules applied at this microcapsule density. This differencecould be explained by an increase in binding strength due to additionalavidity of the bivalent VHH compared to monovalent VHH, it could also bethat the use of bivalent VHH increases flexibility and spacer length ofthe coupled targeting agents on microcapsules, or a combination of both.

TABLE 6 Surface areas of bound microcapsules to wells coated with potatolectin or unrelated antigen Mono- Blank Surface valent Bivalentunrelated micro- coverage VHH 3E6 VHH 3E6 VHH capsules no coating 100%74.536 66.176 77.014 84.982 potato lectin 100% 415.773 490.546 141.63690.030 potato lectin  20% 307.478 511.303 43.452 44.024 potato lectin 4% 59.377 155.759 19.170 10.599 no coating 100% 72.036 55.841 68.10966.509 unrelated 100% 69.503 45.677 78.205 50.965 antigen unrelated  20%27.742 22.114 30.459 17.831 antigen unrelated  4% 5.011 15.038 19.7556.279 antigen

A leaf disc binding assay was used to evaluate the interaction ofVHH-coupled microcapsules with potato, grass and azalea leaves. ø8 mmleaf discs were sampled from the leaves of potato pot plants (varietyDesirée), from the leaves of greenhouse-grown Lollium perenne and fromthe leaves of azalea pot plants. Leaf discs were washed three times withPBS. Microcapsules containing lambda cyhalothrin or Uvitex were dilutedto appropriate densities in 1% skimmed milk in PBS with 0.05%-TWEEN®-20.Microcapsules were added to the leaf discs and settling of microcapsulesand binding of targeting agents allowed for 1 hour. Unboundmicrocapsules were removed by washing three to five times with PBS with0.05%-TWEEN®-20.

For lambda cyhalothrin microcapsules, a residue analysis was performedto measure lambda cyhalothrin amounts on potato leaf discs. Washed leafdiscs with bound microcapsules were transferred to glass vials andmicrocapsules were dissolved in acetone. Samples were diluted byaddition of hexane containing 0.05% triphenylphosphate as internalstandard. The amount of lambda cyhalothrin was determined by GC/MS-MSanalysis in comparison with calibration solutions. Controls for lambdacyhalothrin microcapsules included blank microcapsules to which no VHHwere coupled and microcapsules to which unrelated VHH were coupled.Based on the results of leaf disc binding assays with lambda cyhalothrinmicrocapsules, it was found that some of the VHH hereof are capable ofbinding and retaining microcapsules to leaf surfaces resulting in a3.3-fold and 2.2-fold increase of amounts of lambda cyhalothrin on leafdiscs compared to blank microcapsules to which no VHH were coupled ormicrocapsules with coupled unrelated VHH, respectively.

Leaf discs with Uvitex microcapsules were analyzed for boundmicrocapsules on a macrozoom microscope system (Nikon). Microcapsuleswere counted using Volocity image analysis software (Perkin Elmer). ADAPI filter was used to visualize Uvitex microcapsules. Controls forUvitex microcapsules included blank microcapsules to which no VHH werecoupled and microcapsules to which unrelated VHH were coupled. Based onthe results of the leaf disc binding assay with Uvitex microcapsules itwas found that some of the VHH (e.g., VHH 3E6) hereof proved capable ofbinding and retaining microcapsules specifically to leaf surfaces.

On potato leaf discs, specific binding of the microcapsules coupled withVHH 3E6, resulted in nine-fold more microcapsules bound to leaf surfacescompared to blank microcapsules and in six-fold more microcapsules boundto leaf surfaces compared to microcapsules coupled with unrelated VHH,as shown in FIG. 4. On grass leaf discs, specific binding ofmicrocapsules coupled with VHH 3E6 resulted in three-fold moremicrocapsules bound to leaf surfaces compared to blank microcapsules andin two-fold more microcapsules bound to leaf surfaces compared tomicrocapsules coupled with unrelated VHH. On azalea leaf discs, nospecific binding of microcapsules coupled with VHH 3E6 could beobserved, which entirely resembles the plant-species related bindingspecificity of the VHH as demonstrated in Example 3.

A titration experiment was performed to investigate what dilution factorof microcapsules with specific VHH corresponds to an application ofmicrocapsules to which no VHH were coupled to obtain similar amounts ofmicrocapsules after an identical treatment. Two-fold serial dilutions ofmicrocapsules were prepared and leaf disc binding was analyzed on potatoleaf discs for these dilution series. From the dosing experiment it wascalculated that an eight-fold lower concentration of microcapsules withspecific VHH resulted in similar amounts of microcapsules specificallybound to the leaf discs compared to non-functionalized microcapsules asshown in FIG. 5. From this experiment, it will be clear that ameaningful reduction of a suitable dose of an agrochemical can beachieved, by coupling one of the VHH hereof, to a microcarriercontaining the agrochemical.

Example 6: Deposition and Retention of Targeting Agent-CoupledMicrocapsules on Intact Living Plant Surface

Effects on deposition and retention of carriers with coupled targetingagents were investigated in experiments with whole potato pot plants(variety Desirée) grown in greenhouses. Microcapsules coupled withspecific VHH, coupled with unrelated control VHH, or blank microcapsuleswere applied to multiple whole compound leaves from different plants. Intotal 15 plants were used for different treatments. Microcapsulesuspensions were calculated to apply 6.4% coverage of microcapsules onleaf surfaces. Compound leaves were submerged in microcapsulesuspensions in the same way as for microcapsule leaf disc binding assays(see above) with the modification that settling of microcapsules andbinding of VHH was allowed for only 15 minutes. Plants were allowed todry up for 1 hour after application of microcapsules. One of each pairof opposite leaves from within each compound leaf was sampled andanalyzed without any further treatment.

The effects of specific VHH coupled to microcapsules on microcapsuledeposition could be analyzed with these leaves from differentapplications. The whole plants missing only the sampled leaves weretreated further to investigate the effect of specific VHH coupled tomicrocapsules on retention after a rainfall event and the combinedeffects of deposition and retention. A rain simulation with finedroplets (SSCOTFVS2 nozzle type) of 1 L/m2 in 45 seconds was used toinvestigate retention effects. The opposite leaves of already sampledleaves were sampled after the rain simulation. Whole leaves with Uvitexmicrocapsules were analyzed for bound microcapsules on a macrozoommicroscope system (Nikon). Microcapsules were counted using Volocityimage analysis software (Perkin Elmer). A DAPI filter was used tovisualize Uvitex microcapsules. From the leaves that were sampled beforethe rainfall event it was calculated that already 2.7-fold moremicrocapsules were deposited for microcapsules with specific targetingagent compared to blank microcapsules. Leaves with microcapsules withunrelated control targeting agent contained only a 0.8 fraction ofmicrocapsules compared to blank microcapsules. This shows that specificVHH already have a beneficial effect on the deposition of microcapsuleson plants. On average 69 (±8)% of microcapsules with specific VHH wasretained after the rainfall event while only 35 (±17)% and 39 (±4)% ofmicrocapsules was retained for microcapsules coupled with unrelatedcontrol VHH and blank microcapsules, respectively. The combination ofeffects of deposition and retention resulted in five-fold and 0.9-foldin the amount of microcapsules on leaves on whole plants formicrocapsules with specific VHH or unrelated control VHH, compared toblank microcapsules, respectively.

From this experiment, it will be clear that specific VHH are superiortargeting agents that enable delivery and specific binding of carriersto whole intact living plants. As a consequence of improved depositionand improved retention targeting agents hereof coupled to carrierscontaining an agrochemical or a combination of agrochemicals hold greatpromise to deliver the agrochemicals specifically to plant surfaces andhereby either increase amounts of the agrochemicals deposited on theplant surface, or enable reduced application rates while maintainingsimilar efficacy, or enable reduced application frequencies whilemaintaining similar efficacy or enable improved rainfastness of theagrochemicals or induce a certain specificity for the agrochemicals orany combination of the foregoing.

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1.-50. (canceled)
 51. A targeting agent comprising: at least one bindingdomain able to bind at least one binding site on the surface of at leastone intact living plant or plant part; wherein the binding domain is aVHH; wherein the targeting agent is coupled to an agrochemical, acarrier comprising an agrochemical, or a carrier bound to anagrochemical; and wherein the targeting agent is able to bind and retainthe agrochemical, the carrier comprising an agrochemical, or the carrierbound to an agrochemical, onto the plant or plant part.
 52. Thetargeting agent of claim 51, wherein the binding domain binds to astructure on the plant or plant part.
 53. The targeting agent of claim52, wherein the structure is selected from the group consisting of atrichome, stomata, lenticel, thorn, spine, root hair, cuticle and waxlayer.
 54. The targeting agent of claim 51, wherein the binding domainbinds to gum Arabic.
 55. The targeting agent of claim 51, wherein thebinding domain binds to a lectin, lectin-like domain, extensin, orextensin-like domain.
 56. The targeting agent of claim 51 wherein theVHH has two disulfide bridges.
 57. The targeting agent of claim 51,wherein the VHG is selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQID NO:42.
 58. The targeting agent of claim 51, wherein the agrochemicalis selected from the group consisting of an insecticide, herbicide,fungicide, fertilizer, growth regulator, micro-nutrient, safener,pheromone, repellant, insect bait, and nucleic acid.
 59. The targetingagent of claim 51, wherein the agrochemical is selected from the groupconsisting of glyphosate, paraquat, metolachlor, acetochlor, mesotrione,2,4-D, atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin,picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron,bensulfuron, imazetapyr, dicamba, imidacloprid, thiamethoxam, fipronil,chlorpyrifos, deltamethrin, lambda cyhalothrin, endosulfan,methamidophos, carbofuran, clothianidin, cypermethrin, abamectin,diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin,azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid,fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copperfungicides, trifloxystrobin, prothioconazole, difenoconazole,carbendazim, propiconazole, thiophanate, sulphur, and boscalid.
 60. Thetargeting agent of claim 51, wherein the carrier is selected from thegroup consisting of a microcapsule, microsphere, polymer particle,particle made from artificially lignified cellulose, composite gelparticle, weak ionic resin particle, microbial cell, and fragment of anythereof.
 61. The targeting agent of claim 51, wherein the binding domainbinds to a leaf of the plant.
 62. The targeting agent of claim 51,wherein the dissociation constant of the binding domain to the bindingsite is lower than 10⁻⁵ M.
 63. The targeting agent of claim 51, whereinthe binding domain remains bound to the binding site under conditionscomprising: pH range from pH 2 to 11; and temperature range from 4 to70° C.
 64. A VHH coupled to an agrochemical, a carrier coupled to anagrochemical, or a carrier bound to an agrochemical.
 65. The VHH ofclaim 64, comprising a carrier, wherein the carrier is selected from thegroup consisting of a microcapsule, microsphere, polymer particle,particles made from artificially lignified cellulose, composite gelparticle, weak ionic resin particle, microbial cell, and fragment of anythereof.
 66. The VHH of claim 64, wherein the agrochemical is selectedfrom the group consisting of glyphosate, paraquat, metolachlor,acetochlor, mesotrione, 2,4-D, atrazine, glufosinate, sulfosate,fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil,clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba,imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambdacyhalothrin, endosulfan, methamidophos, carbofuran, clothianidin,cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin,tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb,cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil,copper fungicides, trifloxystrobin, prothioconazole, difenoconazole,carbendazim, propiconazole, thiophanate, sulphur, and boscalid.
 67. TheVHH of claim 64, wherein the VHH is selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, and SEQ ID NO:42.
 68. A method for using the targeting agent ofclaim 51 to deliver an agrochemical or a combination of agrochemicals toa plant or plant part, the method comprising: applying the targetingagent to the plant or plant part.
 69. A method of using the targetingagent of claim 51 to protect a plant or plant part and/or to modulatethe viability, growth or yield of a plant or plant part and/or tomodulate gene expression in a plant or plant part, the methodcomprising: applying the targeting agent to the plant or plant part. 70.A method of using the targeting agent of claim 51 to protect a plantpart post-harvest, the method comprising: applying the targeting agentto the plant part.